Scarecrow gene

ABSTRACT

The structure and function of a regulatory gene, SCARECROW (SCR), is described. The SCR gene is expressed specifically in root progenitor tissues of embryos, and in roots and stems of seedlings and plants. SCR expression controls cell division of certain cell types in roots and affects the organization of root and stem tissues, and affects gravitropism of aerial structures. The invention relates to the SCARECROW (SCR) gene, SCR gene products, (including but not limited to transcriptional products such as mRNAs, antisense, and ribozyme molecules, and translational products such the SCR protein, polypeptides, peptides and fusion proteins related thereto), antibodies to SCR gene products, SCR promoters and regulatory regions and the use of the foregoing to improve agronomically valuable plants.

This application is a continuation-in-part of application Ser. No.08/638,617, filed Apr. 26, 1996, now abandoned the disclosure of whichis incorporated by reference in its entirety.

This invention was made with government support under grant number:GM43778 awarded by the National Institute of Health. The government mayhave certain rights in the invention.

TABLE OF CONTENTS

1. INTRODUCTION

2. BACKGROUND OF THE INVENTION

2.1. Root Development

2.2. Genes Regulating Root Structure

2.3. Geortropism

3. SUMMARY OF THE INVENTION

3.1. Definitions

4. BRIEF DESCRIPTION OF THE FIGURES

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. SCR Genes

5.1.1. Isolation of SCR Genes

5.1.2. Expression of SCR Gene Products

5.1.3. Antibodies to SCR Proteins and Polypeptides

5.1.4. SCR Gene of Gene Products as Markers for Qualitative Trait Loci

5.2. SCR Promoters

5.2.1. Cis-Regulatory Elements or SCR Promoters

5.2.2. SCR Promoter-Driven Expression Vectors

5.3. Production of Transgenic Plants and Plant Cells

5.3.1. Transgenic Plants that Ectopically Express SCR

5.3.2. Transgenic Plants that Suppress Endogenous SCR Expression

5.3.3. Transgenic Plants that Express a Transgene Controlled by the SCRPromoter

5.3.4. Screening of Transformed Plants for Those Having Desired AlteredTraits

EXAMPLE 1: Arabidopsis SCR Gene

6.1 Material and Methods

6.1.1. Plant Culture

6.1.2. Genetic Analysis

6.1.3. Mapping

6.1.4. Phenotypic Analysis

6.1.5. Molecular Techniques

6.1.6. In Situ Hybridization

6.2. Results

6.2.1. Characterization of the SCR Phenotype.

6.2.2. Characterization of Cell Identify in SCR Roots

6.2.3. Molecular Cloning of the SCR Gene

6.2.4. The SCR Gene has Motifs that Indicate it is a TranscriptionFactor

6.2.5. SCR is a Member of a Novel Protein Family

6.2.6. SCR is Expressed in the Cortex/Endodermal Initials and in theEndodermis

6.3. Discussion

6.3.1. The SCR Gene Regulates an Asymmetric Division Required for RootRadial Organization

6.3.2. SCR Involvement in Cell Specification of Cell Division

6.3.3. A Role for SCR in Embryonic Development

6.3.4. Tissue-Specific Expression of SCR is Regulated at theTranscriptional Level

6.3.5. A new Family of Transcriptional Regulators

7. EXAMPLE 2: Enhancer Trap Analysis of Root Development

7.1. Material and Methods

7.1.1. Plant Growth Conditions

7.1.2. Histology and Gus Staining

7.1.3. Construction of Enhancer Trap Lines

7.2. Results

7.2.1. Differential in the LRP

7.2.2. Marker Lines

7.2.3. ET199 Provides Evidence for the Role of SCR in Plant Development

8. EXAMPLE 3: Activity of Arabidopsis SCR Promoter in Transgenic Roots

9. EXAMPLE 4: Isolation SCR Sequences Using PRC-Cloning Strategy

10. EXAMPLE 5: Expression Pattern of Maize ZCR Gene in Root Tissue

11. EXAMPLE 6: Expression Pattern of ZCR Gene in Soybean Roots and RootNodules

12. EXAPMPLE 7: SCR Expression Affects Gravitropism of Aerial Structures

13. Deposit of Microorganisms

1. INTRODUCTION

The present invention generally relates to the SCARECROW (SCR) genefamily and their promoters. The invention more particularly relates toectopic expression of members of the SCARECROW gene family in transgenicplants to artificially modify plant structures. The invention alsorelates to utilization of SCARECROW promoter for tissue and organspecific expression of heterologous gene products.

2. BACKGROUND OF THE INVENTION

Asymmetric cell divisions, in which a cell divides to give two daughterswith different fates, play an important role in the development of allmulticellular organisms. In plants, because there is no cell migration,the regulation of asymmetric cell divisions is of heightened importancein determining organ morphology. In contrast to animal embryogenesis,most plant organs are not formed during embryogenesis. Rather, cellsthat form the apical meristems are set aside at the shoot and rootpoles. These reservoirs of stem cells are considered to be the source ofall post-embryonic organ development in plants. A fundamental questionin developmental biology is how meristems function to generate plantorgans.

2.1. Root Development

Root organization is established during embryogenesis. This organizationis propagated during postembryonic development by the root meristem.Following germination, the development of the postembryonic root is acontinuous process, a series of initials or stem cells continuouslydivide to perpetuate the pattern established in the embryonic root(Steeves & Sussex, 1972, Patterns in Plant Development, EnglewoodCliffs, N.J.: Prentice-Hall, Inc.).

Due to the organization of the Arabidopsis root it is possible to followthe fate of cells from the meristem to maturity and identify theprogenitors of each cell type (Dolan et al., 1993, Development119:71-84). The Arabidopsis root is a relatively simple and wellcharacterized organ. The radial organization of the mature tissues inthe Arabidopsis root has been likened to tree rings with the epidermis,cortex, endodermis and pericycle forming radially symmetric cell layersthat surround the vascular cylinder (FIG. 1A). See also Dolan et al.,1993, Development 20 119:71-84. These mature tissues are derived fromfour sets of stem cells or initials: i) the columella root cap initial;ii) the pericycle/vascular initial; iii) the epidermal/lateral root capinitial; and iv) the cortex/endodermal initial (Dolan et al., 1993,Development 119:71-84). It has been shown that these initials undergoasymmetric divisions (Scheres et al., 1995, Development 121:53-62). Thecortex/endodermal initial, for example, first divides anticlinally (in atransverse orientation) (FIG. 1B). This asymmetric division producesanother initial and a daughter cell. The daughter cell, in turn, expandsand then divides periclinally (in the longitudinal orientation) (FIG.1B). This second asymmetric division produces the progenitors of theendodermis and the cortex cell lineages (FIG. 1B). 2.2. Genes RegulatingRoot Structure

Mutations that disrupt the asymmetric divisions of the cortex/endodermalinitial have been identified and characterized (Benfey et al., 1993,Development 119:57-70; Scheres et al., 1995, Development 121:53-62).short-root (shr) and scarecrow (scr) mutants are missing a cell layerbetween the epidermis and the pericycle. In both types of mutants thecortex/endodermal initial divides anticlinally, but the subsequentpericlinal division that increases the number of cell layers does nottake place (Benfey et al., 1993, Development 119:57-70; Scheres et al.,1995, Development 121:53-62). The defect is first apparent in the embryoand it extends throughout the entire embryonic axis which includes theembryonic root and hypocotyl (Scheres et al., 1995, Development121:53-62). This is also true for the other radial organization mutantscharacterized to date, suggesting that radial patterning that occursduring embryonic development may influence the post-embryonic patterngenerated by the meristematic initials (Scheres et al., 1995,Development 121:53-62).

Characterization of the mutant cell layer in shr indicated that twoendodermal-specific markers were absent (Benfey et al., 1993,Development 119:57-70). This provided evidence that the wild-type SHRgene may be involved in specification of endodermis identity.

2.3. Geotropism

In plants, the capacity for gravitropism has been correlated with thepresence of amyloplast sedimentation. See, e.g., Volkmann and Sievers,1979, Encyclopedia Plant Physiol., N.S. vol 7, pp. 573-600; Sack, 1991,Intern. Rev. Cytol. 127:193-252; Björkmann, 1992, Adv. Space Res.12:195-201; Poff et al., in The Physiology of Tropisms, Meyerowitz &Somerville (eds); Cold Spring Harbor Laboratory Press, Plainview, N.Y.(1994) pp. 639-664; Barlow, 1995, Plant Cell Environ. 18:951-962.Amyloplast sedimentation only occurs in cells in specific locations atdistinct developmental stages. That is, when and where sedimentationoccurs is precisely regulated (Sack, 1991, Intern. Rev. Cytol.127:193-252). In roots, amyloplast sedimentation only occurs in thecentral (columella) cells of the rootcap; as these cells mature intoperipheral cap cells, the amyloplasts no longer sediment (Sack & Kiss,1989, Amer. J. Bot. 76:454-464; Sievers & Braun, in The Root Cap:Structure and Function, Wassail et al. (eds.), New York: M. Dekker(1996) pp. 31-49). In stems of many plants, including Arabidopsis,amyloplast sedimentation occurs in the starch sheath (endodermis)especially in elongating regions of the stem (von Guttenberg, DiePhysiologischen Scheiden, Handbuch der Pflanzenanatomie;

K. Linsbauer (ed.), Berlin: Gebruder Borntraeger, vol. 5 (1943) p. 217;Sack, 1987, Can. J. Bot. 65:1514-1519; Sack, 1991, Intern. Rev. Cytol.127:193-252; Caspar & Pickard, 1989, Planta 177:185-197; Volkmann etal., 1993,J. Pl. Physiol. 142:710-6).

Gravitropic mutants have been studied for evidence that proves the roleof amyloplast sedimentation in gravity sensing. However, manygravitropic mutations affect downstream events such as auxin sensitivityor metabolism (Masson, 1995, BioEssays 17:119-127). Other mutations seemto affect gene products that process information from gravity sensing.For example, the lazy mutants of higher plants and comparable mutants inmosses can clearly sense and respond to gravity, but the mutationsreverse the normal polarity of the gravitropic response (Gaiser & Lomax,1993, Plant Physiol. 102:339-344; Jenkins et al., 1986, Plant CellEnviron 9:637-644). Other mutations appear to affect gravitropism ofspecific organs. For example, sgr mutants have defective shootgravitropism (Fukaki et al., 1996, Plant Physiol. 110:933-943; Fukaki etal., 1996, Plant Physiol. 110:945-955; Fukaki et al., 1996, Plant Res.109:129-137).

Citation or identification of any reference herein shall not beconstrued as an admission that such reference is available as prior artto the present invention.

3. SUMMARY OF THE INVENTION

The structure and function of a regulatory gene, SCARECROW (SCR), isdescribed. The SCR gene is expressed specifically in root progenitortissues of embryos, and in certain tissues of roots and stems. SCRexpression controls cell division of certain cell types in roots, andaffects the organization of root and stem. The invention relates to theSCARECROW (SCR) gene (which encompasses the Arabidopsis SCR gene and itsorthologs and paralogs), SCR gene products, (including but not limitedto transcriptional products such as mRNAs, antisense and ribozymemolecules, and translational products such as the SCR protein,polypeptides, peptides and fusion proteins related thereto), antibodiesto SCR gene products, SCR regulatory regions and the use of theforegoing to improve agronomically valuable plants.

The invention is based, in part, on the discovery, identification andcloning of the gene responsible for the scarecrow phenotype. In contrastto the prevailing view that the SCR gene was likely to be involved inthe specification of endodermis, the inventors have determined that themutant cell layer in roots of scr mutants has differentiatedcharacteristics of both cortex and endodermis. This is consistent with arole for SCR in the regulation of the asymmetric cell division ratherthan in specification of the identity of either cortex or endodermis.The inventors have also determined that SCR expression affects thegravitropism of plant aerial structures such as the stem.

One aspect of the invention relates to the heterologous expression ofSCR genes and related nucleotide sequences, and specifically theArabidopsis SCR genes, in stably transformed higher plant species.Modulation of SCR expression levels can be used to advantageously modifyroot and aerial structures of transgenic plants and enhance theagronomic properties of such plants.

Another aspect of the invention relates to the use of promoters of SCRgenes, and specifically the use of Arabidopsis SCR promoter to controlthe expression of protein and RNA products in plants. Plant SCRpromoters have a variety of uses, including but not limited toexpressing heterologous genes in the embryo, root, root nodule, and stemof transformed plants.

The invention is illustrated by working examples described infra whichdemonstrate the isolation of the Arabidopsis SCR gene using insertionmutagenesis. More specifically, T-DNA tagging of genomic and cDNA clonesof the Arabidopsis SCR gene are described. Additional working examplesinclude the isolation of SCR sequences from plant genomes using PCRamplification in combination with screening of genomic libraries, andheterologous gene expression in transgenic plants using SCR promoterexpression constructs.

Structural analysis of the deduced amino acid sequence of ArabidopsisSCR protein indicates that SCR encodes a transcription factor. Northernanalysis, in situ hybridization analysis and enhancer trap analysis showhighly localized expression of Arabidopsis SCR in embryos and roots.Genetic analysis shows SCR expression also affects gravitropism ofaerial structures (e.g., stems). This indicates that SCR is alsoexpressed in those structures.

Computer analysis of the deduced amino acid sequence of Arabidopsis SCRprotein with those of Expressed Sequence Tag (EST) sequences in GenBankreveals the existence of at least thirteen SCR genes in Arabidopsis, oneSCR gene in maize, four SCR genes in rice, and one SCR gene in Brassica.A further aspect of the invention relates to the use of such ESTsequences to obtain larger and/or complete clones of the correspondingSCR gene.

The various embodiments of the claimed invention presented herein are bythe way of illustration and are not meant to limit the invention.

3.1. Definitions

As used herein, the terms listed below will have the meanings indicated.

35S=cauliflower mosaic virus promoter for the 35S transcript

cDNA=complementary DNA

cis-regulatory element=A promoter sequence 5′ upstream of the TATA boxthat confers specific regulatory response to a promoter containing suchan element. A promoter may contain one or more cis-regulatory elements,each responsible for a particular regulatory response

coding sequence=sequence that encodes a complete or partial gene product(e.g., a complete protein or a fragment thereof)

DNA=deoxyribonucleic acid

EST=expression tagged

functional portion=a functional portion of a promoter is any portion ofa promoter that is capable of causing transcription of a linked genesequence, e.g., a truncated promoter

gene fusion=a gene construct comprising a promoter operably linked to aheterologous gene, wherein said promoter controls the transcription ofthe heterologous gene

gene product=the RNA or protein encoded by a gene sequence

gene sequence=sequence that encodes a complete gene product (e.g., acomplete protein)

GUS=1,3-β-Glucuronidase

gDNA=genomic DNA

heterologous gene=In the context of gene constructs, a heterologous genemeans that the gene is linked to a promoter that said gene is notnaturally linked to. The heterologous gene may or may not be from theorganism contributing said promoter. The heterologous gene may encodemessenger RNA (mRNA), antisense RNA or ribozymes

homologous promoter=a native promoter of a gene that selectivelyhybridizes to the sequence of a SCR gene described herein

mRNA=messenger RNA

operably linked=A linkage between a promoter and gene sequence such thatthe transcription of said gene sequence is controlled by said promoter

ortholog=related gene in a different plant (e.g., maize ZCARECROW geneis an ortholog of the Arabidopsis SCR gene)

paralog=related gene in the same plant (e.g., Arabidopsis SRPa1 is aparalog of Arabidopsis SCR gene)

RNA=ribonucleic acid

RNase=ribonuclease

SCR=SCARECROW gene or gene product, encompasses (italic) SCR and ZCRgenes and their orthologs and paralogs

SCR=SCARECROW protein

scr=scarecrow mutant (e.g., scr1) (lower case)

ZCR=maize ZCARECROW gene, a paralog of, for example, the Arabidopsis SCRgene

SCR protein means a protein containing sequences or a domainsubstantially similar to one or more motifs (i.e., Motif I-VI),preferably MOTIF III (amino acid residues 373-435 of SEQ ID NO:2)(VHIID), (amino acid residues 8-12 of SEQ ID NO:12) of Arabidopsis SCRprotein as shown in FIGS. 13A-F and FIGS. 15A-S. SCR proteins includeSCR ortholog and paralog proteins having the structure and activitiesdescribed herein.

SCR polypeptides and peptides include deleted or truncated forms of theSCR protein, and fragments corresponding to the SCR motifs describedherein.

SCR fusion proteins encompass proteins in which the SCR protein or anSCR polypeptide or peptide is fused to a heterologous protein,polypeptide or peptide.

SCR gene, nucleotides or coding sequences means nucleotides, e.g., gDNAor cDNA encoding SCR protein, SCR polypeptides or peptides, or SCRfusion proteins.

SCR gene products include transcriptional products such as mRNAs,antisense and ribozyme molecules, as well as translational products ofthe SCR nucleotides described herein including but not limited to theSCR protein, polypeptides, peptides and/or SCR fusion proteins.

SCR promoter means the regulatory region native to the SCR gene in avariety of species, which promotes the organ and tissue specific patternof SCR expression described herein.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B. Schematic of Arabidopsis root anatomy. FIG. 1A. Transversesection showing the four tissues, epidermis, cortex, endodermis andpericycle that surround the vascular tissue. In the longitudinalsection, the epidermal/lateral root cap initials and thecortex/endodermal initials are shown at the base of their respectivecell files. FIG. 1B. Schematic of division pattern of thecortex/endodermal initial. The initial expands then divides anticlinallyto reproduce itself and a daughter cell. The daughter then dividespericlinally to produce the progenitors of the endodermis and cortexcell lineages. Abbreviations: C, cortex; Da, daughter cell; E,endodermis; In, initial.

FIGS. 2A-F. Phenotype of scr mutant plants. FIG. 2A. Shown left to rightare 12-day scr-2, scr-1 and wild-type seedlings grown vertically onnutrient agar medium. FIG. 2B. 21-day scr-2 mutant plants in soil. FIG.2C. Transverse section through primary root of 7-day scr-2. FIG. 2D.Transverse section through primary root of 7-day wild-type (WT). FIG.2E. Transverse section through lateral root of 12-day scr-1 mutantseedling. FIG. 2F. Transverse section through root regenerated fromscr-1 callus. Bar, 50 μm. Abbreviations: C, cortex; En, endodermis; Ep,epidermis; M, mutant cell layer; P, pericycle; V, vascular tissue.

FIGS. 3A-F. Characterization of the cellular identity of the mutant celllayer. FIG. 3A. Endodermis-specific Casparian band staining oftransverse sections through the primary root of 7-day scr-1 mutant.(Note: the histochemical stain also reveals xylem cells in the vascularcylinder.) FIG. 3B. Casparian band staining of transverse sectionsthrough the primary root of 7-day wild-type (WT). FIG. 3C.Immunostaining with the endodermis (and a subset of vascular tissue)specific JIM13 monoclonal antibodies on transverse root sections ofscr-2 mutant. FIG. 3D. Immunostaining with JIM13 monoclonal antibodieson transverse root sections of WT. FIG. 3E. Immunostaining with the JIM7monoclonal antibody that stains all cell walls on transverse rootsections of scr-2 mutant. FIG. 3F. Immunostaining with JIM7 monoclonalantibodies on transverse root sections of WT. Bar, 25 μm. Abbreviationsare same as those for description of FIGS. 2A-2F and: Ca, casparianstrip.

FIGS. 4A-F. Immunostaining. FIG. 4A. Immunostaining with the cortex (andepidermis) specific CCRC-M2 monoclonal antibodies on transverse rootsections of scr-1 mutant. FIG. 4B. Immunostaining with CCRC-M2antibodies on transverse root sections of scr-2 mutant. FIG. 3C.Immunostaining with CCRC-M2 antibodies on transverse root sections ofwild-type (WT). FIG. 4D. Immunostaining with the CCRC-M1 monoclonalantibodies (specific to a cell wall epitope found on all cells) ontransverse root sections of scr-1. FIG. 4E. Immunostaining with CCRC-M1antibodies on transverse root sections of scr-2. FIG. 4F. Immunostainingwith CCRC-M1 antibodies on transverse root sections of WT. Bar, 30 μm.Abbreviations are same as those for description of FIGS. 2A-2F.

FIGS. 5A-E. Structure of the Arabidopsis SCARECROW gene. FIG. 5A.Nucleic acid sequence and deduced amino acid sequence of the ArabidopsisSCR genomic region (SEQ ID NO:1) and (SEQ ID NO:2), respectively.Regulatory sequences including: (i) TATA box, (ii) ATG start codon, and(iii) potential polyadenylation sequence are underlined. Within thededuced amino acid sequence homopolymeric repeats are underlined. FIG.5B. Schematic diagram of genomic clone indicating possible functionalmotifs, T-DNA insertion sites and subclones used as probes.Abbreviations: Q,S,P,T, region with homopolymeric repeats of these aminoacids; b, region with similarity to the basic region of bZIP factors; Iand II, regions with leucine heptad repeats; E, acidic region. FIG. 5C.Comparison of the charged region found in Arabidopsis SCR protein withthat found in bZIP transcription factors, SCR bZIP-like domain (SEQ IDNO:3), GCN4 (SEQ ID NO:4), TGA1 (SEQ ID NO:5), C-Fos (SEQ ID NO:6),c-JUN (SEQ ID NO:7), CREB (SEQ ID NO:8), Opaque-2 (SEQ ID NO:9), OBF2(SEQ ID NO:10), RAF-1 (SEQ ID NO:11). FIG. 5D. Translations of ESTclones encoding putative peptide having similarities to the VHIID domainregion of Arabidopsis SCR protein (SEQ ID NO:12), F13896 (SEQ ID NO:13),Z37192 (SEQ ID NO:14), and Z25645 (SEQ ID NO:15) are from Arabidopsis,T18310 (SEQ ID NO:17) is from maize and D41474 (SEQ ID NO:16) is fromrice. FIG. 5E. The deduced amino acid sequence of the ArabidopsisSCARECROW gene (SEQ ID NO:2).

FIGS. 6A-B. Expression of the Arabidopsis SCARECROW gene. FIG. 6A.Northern blot of total RNA from wild-type siliques (Si), roots (R),leaves (L) and whole seedlings (Sd) hybridized with Arabidopsis SCRprobe a and with a probe from the Arabidopsis glutamine dehydrogenase(GDH) gene (Melo-oliveira et al., 1996, Proc. Natl. Acad. Sci. USA93:4718-4723) as a control for RNA integrity. (GDH expression is lowerin siliques than in vegetative tissues.) The 1.6 kb band corresponds tothe GDH gene and the approximately 2.5 kb band corresponds to SCR.Ribosomal RNA is shown as a loading control. FIG. 6B. Northern blot ofArabidopsis wild-type, scr-1 and scr-2 total RNA, probed withArabidopsis SCR probe “a” corresponding to a cDNA sequence shown in FIG.5B, and with the GDH probe. In scr-2 mutant additional bands of 4.1 kband 5.0 kb were detected.

FIGS. 7A-G. In situ hybridization and enhancer trap analyses ofArabidopsis SCR expression. FIG. 7A. SCR RNA expression detected by insitu hybridization of SCR antisense probe to a longitudinal sectionthrough the root meristem. FIG. 7B. In situ hybridization of SCRantisense probe to a transverse section in the meristematic region. FIG.7C. In situ hybridization of SCR antisense probe to late torpedo stageembryo. FIG. 7D. Negative control in situ hybridization using a SCRsense probe to a longitudinal section through the root meristem. FIG.7E. GUS expression in a whole mount in the enhancer trap line, ET199 inprimary root tip. FIG. 7F. GUS expression in the ET199 line intransverse root section in the meristematic region. FIG. 7G. GUSexpression in ET199 detected in a section through the root meristem. GUSexpression is observed in the cortex/endodermal initial, and in thefirst cell in the endodermal cell lineage but not in the first cell ofthe cortex lineage. Expression in two endodermal layers is observedhigher up in the root because the section was not median at that point.Bar, 50 μm. Abbreviations are same as those in the description of FIGS.2A-2F.

FIG. 8. Partial nucleotide sequence (SEQ ID NO:18) and deduced aminoacid sequence (SEQ ID NO:19) of the Arabidopsis SRPa4 gene.

FIG. 9. Partial nucleotide sequence (SEQ ID NO:20) and deduced aminoacid sequence (SEQ ID NO:21) of the Arabidopsis SRPa3 gene.

FIG. 10. Partial nucleotide sequence (SEQ ID NO22) of the ArabidopsisSRPa1 gene.

FIG. 11A. Nucleotide sequence (SEQ ID NO:24) and deduced amino acidsequence (SEQ ID NO:25) of the maize Zm-Scl1 fragment.

FIG. 11B. Partial nucleotide sequence (SEQ ID NO:25) and deduced aminoacid sequence (SEQ ID NO:26) of the maize SRPm1 gene (Zm-Scl2).

FIGS. 12A-B. Nucleotide sequence of rice SRPo3 EST clone. FIG. 12A.Sequence of 5′ end of EST clone (SEQ ID NO:28). FIG. 12B. Sequence of 3′end of EST clone (SEQ ID NO:29).

FIGS. 13A-F. Comparison of the amino acid sequence of members of theSCARECROW family of genes. Conserved Motifs I through VI are indicatedby dashed line above the aligned sequences. Consensus sequences areshown in bold. See Table 1 for the identity and sequence identifiernumber of each of the sequences shown in this Figure. Hu-scr-1=Human SCRparalog (SEQ ID NO:40).

FIG. 14. Restriction map of the approximately 8.8 kb Eco RI insert DNAof lambda clone, t643, containing the Arabidopsis SCR gene. Thelocations of the approximately 5.6 kb HindIII-SacI fragment subcloned inplasmid LIG 1-3/SAC+MoB₂ 1SAC, and the SCR coding region are indicatedbelow the restriction map. The location of the translational initiationsite of the SCR gene is at the Nco I site at the left end of theindicated coding region. The SCR coding sequence begins at thetranslation initiation site and extends approximately 1955 nucleotidesto its right. E. coli DH5α containing plasmid pLIG1-3/SAC+MoB₂ 1SAC, hasthe ATCC accession number 98031.

FIGS. 15A-S. Comparison of the partial and complete amino acid sequencesof several plant members of the SCARECROW family of genes. The aminoacid sequences are aligned in a manner that maximizes amino acidsequence similarity and identity among SCR family members. Each sequenceshown is continuous except where noted otherwise; the dots are insertedbetween two sequence segments in order to align homologous segments. “X”in the middle of a sequence indicates ambiguity in the correspondingnucleotide sequence and, possible termination of the ORF at the “X”residue site. “X” at the end of a sequence indicates termination of theORF at the “X” residue site. The numbering of the amino acid residues isshown at the bottom of each figure and is based on the Arabidopsis SCRamino acid sequence. Conserved Motifs I through VI are indicated by thevarious dashed lines above the figures. The new and old names of thefamily members are shown in FIG. 15A. The sequences of SCR, Tf1 and Tf4are of the complete SCR protein. See Table 1 for the identity and thesequence identifier number of each sequence shown in these figures.

FIGS. 16A-M. The partial nucleotide sequences of several plant membersof the SCARECROW family of genes. “N” indicates an unknown base. SeeTable 1 for the identity and the sequence identifier number of eachsequence shown in these figures.

FIG. 17A. The partial nucleotide sequence (SEQ ID NO:66) of the maizeZCR gene.

FIG. 17B. The partial amino acid sequence (SEQ ID NO:67) of the maizeZCR gene. The underlined sequence shares approximately 80% sequenceidentity with a corresponding sequence of Arabidopsis SCR protein.

FIG. 18. Comparison of the partial amino acid sequences of several SCRortholog sequences amplified from the genomes of carrot, soybean andspruce. The SRPd1 and SRPp1 sequences each were obtained by PCRamplification using a combination of 1F and 1R primers. The SRPg1sequence was obtained by PCR amplification using a combination of 1F andWP primers. The amino acid sequences are aligned in a manner thatmaximizes amino acid sequence identity and similarity amongst thesesequences. Each sequence shown is continuous except where notedotherwise; the dashes are inserted between two sequence segments inorder to allow alignment of homologous segments. “x” in the middle of asequence indicates ambiguity in the corresponding nucleotide sequenceand, possible termination of the ORF or existence of an intron at the“x” residue site. See Table 1 for the identity and the sequenceidentifier number of each sequence shown in this figure.

FIGS. 19A-G. Comparison of promoter activities in transgenic lines androots. Panel a. A stably transformed line containing four copies of theB2 subdomain of the 35S promoter of CaMV upstream of GUS (Benfey et al.,1990). GUS is expressed in the root tip. Panel b. Roots emerging fromcallus transformed with four copies of the B2 subdomain of the 35Spromoter fused to GUS. GUS expression can be seen in the emerging roottips (arrows). Panel c. Higher magnification of a root emerging from thecallus in panel b. GUS is clearly restricted to the root tip. Themorphology of roots regenerated from calli often appears abnormal. Paneld. A transgenic plant regenerated from the calli and roots shown inpanel b. GUS expression in this plants appears to be similar to that ofthe original line shown in panel a. Panel e. ET199, a stably transformedline that contains an enhancer trapping construct with a minimalpromoter fused to the GUS coding region inserted 1 kb upstream from theSCR coding region. GUS expression is primarily in the endodermal layerof the root. Panel f. Roots emerging from calli transformed with the SCRpromoter::GUS construct. Expression of the GUS gene appears to belimited to an internal layer (arrows). Panel g. SCR promoter::GUStransformed root in liquid culture. Roots shown in panel f were excisedand transferred to liquid cultures. GUS expression is primarily found inthe endodermal layer as in ET199. The expression of GUS in the quiescentcenter, as seen here, is also sometimes observed in ET199. Bar, 50 μm.

FIGS. 20A-B. Analysis of SCR promoter activity in the scr mutantbackground. Panel a. Roots emerging from scr calli transformed with theSCR promoter::GUS construct. Roots regenerated from scr calli are veryshort. GUS expression appears to be limited to an internal layer of theroot (arrows). Panel b. Root regenerated from transformed scr calli andtransferred to liquid culture. The scr phenotype, a single layer betweenthe epidermis and pericycle, is easily seen. GUS expression is limitedto this mutant layer. E, Epidermis. M, Mutant Layer. P, Pericycle. Bar,50 μm.

FIGS. 21A-F. Molecular Complementation of the scr mutant. Panels a, cand e. scr transformed with the SCR promoter::GUS construct. Panels b, dand f. scr transformed with the SCR promoter::SCR coding regionconstruct. Panels a and b. Roots emerging from scr calli. Arrows pointto several very short roots among many fine root hairs in the scr callitransformed with the SCR promoter::GUS construct. In contrast, rootsfrom scr calli transformed with the SCR promoter::SCR coding regionconstruct appeared to be wild-type in length, suggesting molecularcomplementation by the transgene. Panels c and d. Transgenic roots inliquid culture. The scr roots transformed with the SCR promoter::GUSconstruct appeared short, while those transformed with the SCRpromoter::SCR coding region construct appeared of wild-type length.Panels e and f. Transverse sections through roots emerging from calli.Whereas there is only a single cell layer between the epidermis andstele in the SCR promoter::GUS transformed root, the radial organizationof the root transformed with the SCR promoter::SCR coding regionappeared identical to wild-type, with both cortex and endodermal layers.E, epidermis. M, mutant layer. C, cortex. En, Endodermis. P, Pericycle.Bar, 50 μm.

FIG. 22. Expression of ZCR in maize root tips. Left Panel. Expression ofZCR is in the endodermal layer and extends down through the region ofthe quiescent center. Right Panel. Higher magnification showingexpression in a single cell layer through the quiescent center.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the SCARECROW (SCR) gene, SCR gene products,including but not limited to transcriptional products such as mRNAs,antisense and ribozyme molecules, and translational products such as theSCR protein, polypeptides, peptides and fusion proteins related thereto;antibodies to SCR gene products; SCR regulatory regions; and the use ofthe foregoing to improve agronomically valuable plants.

In summary, the data described herein show the identification of SCR, agene involved in the regulation of a specific asymmetric division, incontrolling gravitropic response in aerial structures, and incontrolling pattern formation in roots. Sequence analysis shows that theSCR protein has many hallmarks of transcription factors. In situ andmarker line expression studies show that SCR is expressed in thecortex/endodermal initial of roots before asymmetric division occurs,and in quiescent center of regenerating roots. Together, these findingsindicate that SCR gene regulates key events that establish theasymmetric division that generates separate cortex and endodermal celllineages, and that affect tissue organization of roots. Theestablishment of these lineages is not required for cell differentiationto occur, because in the absence of division the resulting cell acquiresmature characteristics of both cortex and endodermal cells. However, itis possible that SCR functions to establish the polarity of the initialbefore cell division, or that it is involved in generating an externalpolarity that has an effect on asymmetric cell division.

Genetic analysis indicates that SCR expression affects gravitropism ofplant stems and hypocotyls. This indicates that SCR is also expressed inthese aerial structures of plants.

The SCR genes and promoters of the present invention have a number ofimportant agricultural uses. The SCR promoters of the invention may beused in expression constructs to express desired heterologous geneproducts in the embryo, root, root nodule, and starch sheath layer instem of transgenic plants transformed with such constructs. For example,SCR promoters may be used to express disease resistance genes such aslysozymes, cecropins, maganins, or thionins for anti-bacterialprotection or the pathogenesis-related (PR) proteins such as glucanasesand chitinases for anti-fungal protection. SCR promoters also may beused to express a variety of pest resistance genes in the aforementionedplant structures and tissues. Examples of useful gene products forcontrolling nematodes or insects include Bacillus thuringiensisendotoxins, protease inhibitors, collagenases, chitinase, glucanases,lectins, and glycosidases.

Gene constructs that express or ectopically express SCR, and theSCR-suppression constructs of the invention may be used to alter theroot and/or stem structure, and the gravitropism of aerial structures oftransgenic plants. Since SCR regulates root cell divisions,overexpression of SCR can be used to increase division of certain cellsin roots and thereby form thicker and stronger roots. Thicker andstronger roots are beneficial in preventing plant lodging. Conversely,suppression of SCR expression can be used to decrease cell division inroots and thereby form thinner roots. Thinner roots are more efficientin uptake of soil nutrients. Since SCR affects gravitropism of aerialstructures, overexpression of SCR may be used to develop “straighter”transgenic plants that are less susceptible to lodging.

Further, SCR gene sequence may be used as a molecular marker for aqualitative trait, e.g., a root or gravitropism trait, in molecularbreeding of crop plants.

For purposes of clarity and not by way of limitation, the invention isdescribed in the subsections below in terms of (a) SCR genes andnucleotides; (b) SCR gene products; (c) antibodies to SCR gene products;(d) SCR promoters and promoter elements; (e) transgenic plants whichectopically express SCR; (f) transgenic plants in which endogenous SCRexpression is suppressed; and (g) transgenic plants in which expressionof a transgene of interest is controlled by SCR promoter.

5.1. SCR Genes

The SCARECROW genes and nucleotide sequences of the invention include:(a) a gene listed below in Table 1 (hereinafter, a gene comprising anyone of the nucleotide sequences shown in FIG. 5A, FIG. 8, FIG. 9, FIG.10, FIGS. 11A-B, FIGS. 12A-B, FIGS. 16A-M, or FIG. 17A, or a segment ofsuch nucleotide sequences), or as contained in the clones describedherein and deposited with the ATCC (see Section 13, infra); (b)nucleotide sequence that encodes a protein comprising any one of theamino acid sequences shown in FIG. 5A, FIG. 5D, FIG. 5E, FIG. 8, FIG. 9,FIGS. 11A-B, FIGS. 13A-F, FIGS. 15A-S, FIG. 17B or FIG. 18 or a segmentof such amino acid sequences, or that is encoded by any one of the genesand/or nucleotide sequences listed by their sequence identifier numbersin Table 1, or any segment of such genes and/or nucleotide sequences, orcontained in any one of the clones described herein and deposited withthe ATCC (see Section 13, infra); (c) any gene comprising nucleotidesequence that hybridizes to the complement of any one of the genesand/or nucleotide sequences listed by their sequence identifier numbersin Table 1, or any segment of such genes and/or nucleotide sequences, oras contained in any one of the clones described herein and depositedwith the ATCC, under highly stringent conditions, e.g., hybridization tofilter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mMEDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M.et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I,Green Publishing Associates, Inc., and John Wiley & sons, Inc., NewYork, at p. 2.10.3) and that encodes a gene product functionallyequivalent to SCR gene product encoded completely or partly by any oneof the genes and/or sequences listed in Table 1 or any segment of suchgenes and nucleotide sequences, or as contained in any one of the clonesdeposited with the ATCC; (d) any gene comprising nucleotide sequencethat hybridizes to the complement of any one of the sequences listed bytheir sequence identifier numbers in Table 1, or any segment of suchnucleotide sequences, or as contained in any one of the clones describedherein and deposited with the ATCC, under less stringent conditions,such as moderately stringent conditions, e.g., washing in 0.2×SSC/0.1%SDS at 42° C. (Ausubel et al., 1989, supra), and which encodes afunctionally equivalent SCR gene product; (e) any gene comprisingnucleotide sequence that hybridizes to the complement of any one of thesequences listed by their sequence identifier numbers in Table 1 or anysegment of such nucleotide sequences, or as contained in any one of theclones described herein and deposited with the ATCC, under the followinglow stringency conditions: pre-hybridization in hybridization solution(HS) containing 43% formamide, 5×SSC, 1% SDS, 10% dextran sulfate, 0.1%sarkosyl, 2% block (Genius kit, Boehringer-Mannheim), followed byhybridization overnight at 30 to 33° C. using as a probe a DNA moleculeof approximately 1.6 kb of SEQ ID NO:1 at a concentration of 20 ng/ml,followed by washing in 2×SSC/0.1% SDS two times for 15 minutes at roomtemperature and then two times at 50° C., and which encodes afunctionally equivalent SCR gene product; and/or (f) any gene comprisingnucleotide sequence that encodes a polypeptide or protein containing theconsensus sequence for SCR (i.e., MOTIF III (amino acid residues 373-435of SEQ ID NO:2) or VHIID) (amino acid residues 8-12 of SEQ ID NO:12)shown in FIGS. 13B-D or a segment of such polypeptide or protein. Thepartial and complete nucleotide and amino acid sequences of SCR genesand encoded proteins and polypeptides included in the invention arelisted in Table 1 below.

TABLE 1 SCR ORTHOLOGS AND PARALOGS SEQ ID NOS New Name Old Name ESTClone¹ Nucleotide³ Amino Acid ARABIDOPSIS SRPa1 1110 Z25645/33772 22 23SRPa2 Tf4 Z34599 — 35* SRPa3 3935 Z37192/1 20 21 N96166 SRPa4 4818F13896/7 18 19 SRPa5 4871 F13949 45 46 SRPa6 12398 R29793 51 52 SRPa73635 T21627 55 56 H76979 N96767 SRPa8 Tf1 T46205 (9468) — 34* N96653(21711) SRPa9 10964 T78186 47 48 T44774 SRPa10 11261 T76483 49 50 SRPa1118652 N37425 53 54 SRPa12 23196 W43803 57 58 W435138 AA042397 SRPa1333/08 T46008 — 41 SCR Scr N.A.²  1⁺  2* RICE SRPo1 713 D15490 — 43 SRPo22504 D40482 — 44 D40607 D40800 D41389 SRPo3 3989 D41474 — 36 SRPo4 11846C20324 — 59 MAIZE SRPm1 18310 T18310 — 37 BRASSICA SRPb1 174 H74669 — 42CARROT SRPd1 N.A. N.A. 60 61 SOYBEAN SRPg1 N.A. N.A. 62 63 SPRUCE SRPp1N.A. N.A. 64 65 ¹Each EST clone is identified by its GenBank accessionnumber. Each EST clone corresponds to a deposit of a cDNA sequence thatmatches a part of the nucleotide sequence of the corresponding SCRortholog or paralog. ²N.A. = not applicable. ³The partial or completenucleotide sequence of the SCR orthologs and paralogs listed here areshown in FIGS. 5A, 8, 9, 10, 11A-B, 12A-B, 16A-M and 17A. ⁺Contains thecomplete coding sequence of Arabidopsis SCR gene. *Contains the completeamino acid sequence of Arabidopsis SRPa2, SRPa8, or SCR protein.

Functional equivalents of the SCR gene product include any plant geneproduct that regulates plant embryo or root development, or, preferably,that regulates root cell division or root tissue organization, oraffects gravitropism of plant aerial structures (e.g., stems andhypocotyls). Functional equivalents of the SCR gene product includenaturally occurring SCR gene products, and mutant SCR gene products,whether naturally occurring or engineered.

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of thenucleotide sequences (a) through (f), in the first paragraph of thissection. Such hybridization conditions may be highly stringent, lesshighly stringent, or low stringency as described above. In instanceswherein the nucleic acid molecules are oligonucleotides (“oligos”),highly stringent conditions may refer, e.g., to washing in 6×SSC/0.05%sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules may act as SCR antisense molecules, useful,for example, in SCR gene regulation and/or as antisense primers inamplification reactions of SCR gene and/or nucleic acid sequences.Further, such sequences may be used as part of ribozyme and/or triplehelix sequences, also useful for SCR gene regulation. Still further,such molecules may be used as components in probing methods whereby thepresence of a SCARECROW allele may be detected.

The invention also includes nucleic acid molecules, preferably DNAmolecules, which are amplified using the polymerase chain reaction underconditions described in Section 5.1.1., infra, and that encode a geneproduct functionally equivalent to a SCR gene product encoded by any oneof the genes and sequences listed in Table 1 or as contained in any oneof the clones described herein and deposited with the ATCC.

The invention also encompasses (a) DNA vectors that contain any of theforegoing gene and/or coding sequences and/or their complements (i.e.,antisense or ribozyme molecules); (b) DNA expression vectors thatcontain any of the foregoing gene and/or coding sequences operativelyassociated with a regulatory element that directs the expression of thegene and/or coding sequences; and (c) genetically engineered host cellsthat contain any of the foregoing gene and/or coding sequencesoperatively associated with a regulatory element that directs theexpression of the gene and/or coding sequences in the host cell. As usedherein, regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression.

The invention also encompasses nucleotide sequences that encode mutantSCR gene products, peptide fragments of the SCR gene product, truncatedSCR gene products, and SCR fusion proteins. These gene products include,but are not limited to, nucleotide sequences encoding mutant SCR geneproducts; polypeptides or peptides corresponding to one or more of theMotifs I-VI as shown in FIGS. 13A-F and FIGS. 15A-S, or the bZIP, VHIID,or leucine heptad domains of the SCR, or portions of these motifs anddomains; truncated SCR gene products in which one or more of the motifsor domains is deleted, e.g., a truncated, nonfunctional SCR lacking allor a portion of the Motifs I-VI as shown in FIGS. 13A-F and FIGS. 15A-S,or the bZIP, VHIID, or leucine heptad domains of the SCR. Nucleotidesencoding fusion proteins may include but are not limited to full lengthSCR, truncated SCR or peptide fragments of SCR fused to an unrelatedprotein or peptide, such as for example, an enzyme, fluorescent protein,or luminescent protein which can be used as a marker.

In particular, the invention includes, for example, fragments of SCRgenes encoding one or more of the following domains as shown in FIG. 5E:amino acids 1-264, 265-283, 287-316, 410-473, 436-473, and 473-653.

In addition to the gene and/or coding sequences described above,homologous SCR genes, and other genes related by DNA sequence, may beidentified and may be readily isolated, without undue experimentation,by molecular biological techniques well known in the art. Morespecifically, such homologs include, for example, paralogs (i.e.,members of the SCR gene family occurring in the same plant) as well asorthologs (i.e., members of the SCR gene family which occur in adifferent plant species) of the Arabidopsis SCR gene.

A specific embodiment of a SCR gene and coding sequence of the inventionis Arabidopsis SCR (FIGS. 5A and 5E). Other specific embodiments includethe various SCR genes and coding sequences listed in Table 1, supra.

Methods for isolating SCR genes and coding sequences are described indetail in Section 5.2, below.

SCR genes share substantial amino acid sequence similarities at theprotein level and nucleotide sequence similarities in their encodinggenes. The term “substantially similar” or “substantial similarity” whenused herein with respect to two amino acid sequences means that the twosequences have at least 75% identical residues, preferably at least 85%identical residues and most preferably at least 95% identical residues.The same term when used herein with respect to two nucleotide sequencesmeans that the two sequences have at least 70% identical residues,preferably at least 85% identical residues and most preferably at least95% identical residues. Determining whether two sequences aresubstantially similar may be carried out using any methodologies knownto one skilled in the art, preferably using computer assisted analysis.For example, the alignments showed herein were initially accomplished bya BLAST search (NCBI using the BLAST network server). The finalalignments of SCR family members were done manually.

Moreover, SCR genes show highly localized expression in embryos and,particularly, roots. Such expression patterns may be ascertained byNorthern hybridizations and in situ hybridizations using antisenseprobes.

5.1.1. Isolation of SCR Genes

The following methods can be used to obtain SCR genes and codingsequences from a wide variety of plants, including but not limited toArabidopsis thaliana, Zea mays, Nicotiana tabacum, Daucus carota, Oryza,Glycine max, Lemna gibba, and Picea abies.

Nucleotide sequences encoding an SCR gene or a portion thereof may beobtained by PCR amplification of plant genomic DNA or cDNA. Useful cDNAsources include “free” cDNA preparations (i.e., the products of cDNAsynthesis) and cloned cDNA in cDNA libraries. Root cDNA preparations orlibraries are particularly preferred.

The amplification may use, as the 5′-primer (i.e., forward primer), adegenerate oligonucleotide that corresponds to a segment of a known SCRamino acid sequence, preferably from the amino-terminal region. The3′-primer (i.e., reverse primer) may be a degenerate oligonucleotidethat corresponds to a distal segment of the same known SCR amino acidsequence (i.e., carboxyl to the sequence that corresponds to the5′-primer). For example, the amino acid sequence of the Arabidopsis SCRprotein (SEQ ID NO:2) may be used to design useful 5′ and 3′ primers.Preferably, the primers corresponds to segments in the Motif III (aminoacid residues 373-435 of SEQ ID NO:2) or VHIID (amino acid residues 8-12of SEQ ID NO:12) domain of SCR protein (see FIGS. 13B-D and FIGS.15K-L). The sequence of the optimal degenerate oligonucleotide probecorresponding to a known amino acid sequence may be determined bystandard algorithms known in the art. See for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., Vol 2 (1989).

Further, for amplification from cDNA sources, the 3′-primer may be anoligonucleotide comprising an 3′ oligo(dT) sequence. The amplificationmay also use as primers nucleotide sequences of SCR genes or codingsequences (e.g., any one of the scr sequences and EST sequences listedin Table 1).

PCR amplification can be carried out, e.g., by use of a Perkin-ElmerCetus thermal cycler and Taq polymerase (Gene Amp™). One can choose tosynthesize several different degenerate primers for use in the PCRreactions. It is also possible to vary the stringency of hybridizationconditions used in priming the PCR reactions, to allow for greater orlesser degrees of nucleotide sequence similarity between the degenerateprimers and the corresponding sequences in the cDNA library. One ofordinary skill in the art will know that the appropriate amplificationconditions and parameters depend, in part, on the length and basecomposition of the primers and that such conditions may be determinedusing standard formulae. Protocols for executing all PCR proceduresdiscussed herein are well known to those skilled in the art, and may befound in references such as Gelfand, 1989, PCR Technology, Principlesand Applications for DNA Amplification, H. A. Erlich, ed., StocktonPress, New York; and Current Protocols In Molecular Biology, Vol. 2, Ch.15, Ausubel et al., eds 1988, New York, Wiley & Sons, Inc.

A PCR amplified sequence may be molecularly cloned and sequenced. Theamplified sequence may utilized as a probe to isolate genomic or cDNAclones of a SCR gene, as described below. This, in turn, will permit thedetermination of a SCR gene's complete nucleotide sequence, includingits promoter, the analysis of its expression, and the production of itsencoded protein, as described infra.

In a preferred embodiment, PCR amplification of SCR gene and/or codingsequences can be carried out according to the following procedure:

Primers:

Forward:

Name: SCR5AII (23-mer, 2 inosines, 64-mix)

A.A. code: HFTANQAI (SEQ ID NO:69)

DNA Sequence: 5′ CAT/C TTT/C ACI GCI AAT/C CAA/G GCN AT 3′ SEQ ID NO:60

Name: SCR5B (29-mer, 1 inosine, 144-mix)

A.A. code: VHIID(L/F)D SEQ ID NO:71

DNA Sequence: 5′ ACGTCTCGA GTI CAT/C ATA/C/T ATA/C/T GAT/C TTN GA 3′ SEQID NO 70

Name: 1F

A.A. code: LQCAEAV SEQ ID NO:73

DNA Sequence: (T/C)TI CA(A/G) TG(T/C GCI GA(A/G) GCN GT SEQ ID NO:72

Reverse:

Name: SCR3AII (23-mer, 2 inosines, 128-mix)

A.A. code: PGGPP(H/N/K)(V/L/F)R′ SEQ ID NO:75

DNA Sequence: 5′ CG/T CCA/C GTG/T TGG IGG ICC NCC NGG 3′ SEQ ID NO:74

Name: 1R

A.A. code: AFQVFNGI SEQ ID NO:77

DNA Sequence: AT ICC (A/G)TT (A/G)AA IAC (C/T)TG (A/G)AA NGC SEQ IDNO:76

Name: 4R

A.A. code: QWPGLFHI SEQ ID NO:79

DNA Sequence: AT (A/G)TG (A/G)AA IA(A/G) NCC IGG CCA (C/T)TG SEQ IDNO:78

I=inosine

N=A/C/G/T

Useful primer combinations include the following:

SCR5AII+SCR3AII; SCR5B+SCR3AII; IF+IR; and IF+4R

PCR:

Reaction mixture (volume 50 μl):

5 μl 10×amplification buffer containing Mg (Boehringer-Mannheim)

1 μl 10 mM dNTP's

1 μl forward primer (stock concentration: 80 pmol/μl)

1 μl reverse primer (80 pmol/μl)

DNA (100-300 ng).

Begin reaction with “hot start” in which the enzyme is added to the mixonly after a brief denaturation at a high temperature (80° C.)

Cycles:

94° C. 30 sec—brief denaturation (to prevent non-specific priming)

80° C. 5 min—apply the enzyme to the tubes (30 tubes/round at maximum)

94° C. 5 min—thorough denaturation

2 times:

94° C. 1 min

64° C. 5 min

72° C. 2 min

2 times:

94° C. 1 min

62° C. 5 min

72° C. 2 min

2 times:

94° C. 1 min

60° C. 5 min

72° C. 2 min

(reduce the annealing temperature 2° C. in every second round), until44° C. is reached after that:

40 times:

94° C. 20 sec

48° C. 1 min

72° C. 2 min

finally, let cool down to 15° C.

A SCR gene coding sequence may also be isolated by screening a plantgenomic or cDNA library using a SCR nucleotide sequence (e.g., thesequence of any of the SCR genes and sequences and EST clone sequenceslisted in Table 1.) as hybridization probe. For example, the whole or asegment of the Arabidopsis SCR nucleotide sequence (FIG. 5A) may beused. Alternatively, a SCR gene may be isolated from such librariesusing as probe a degenerate oligonucleotide that corresponds to asegment of a SCR amino acid sequence. For example, degenerateoligonucleotide probe corresponding to a segment of the Arabidopsis SCRamino acid sequence (FIG. SE) may be used.

In preparation of cDNA libraries, total RNA is isolated from planttissues, preferably roots. Poly(A)+ RNA is isolated from the total RNA,and cDNA prepared from the poly(A)+ RNA, all using standard procedures.See, for example, Sambrook et al., Molecular Cloning: A LaboratoryManual, 2d ed., Vol. 2 (1989). The cDNAs may be synthesized with arestriction enzyme site at their 3′-ends by using an appropriate primerand further have linkers or adaptors attached at their 5′-ends tofacilitate the insertion of the cDNAs into suitable cDNA cloningvectors. Alternatively, adaptors or linkers may be attached to the cDNAsafter the completion of cDNA synthesis.

In preparation of genomic libraries, plant DNA is isolated and fragmentsare generated, some of which will encode parts of the whole SCR protein.The DNA may be cleaved at specific sites using various restrictionenzymes. Alternatively, one may use DNase in the presence of manganeseto fragment the DNA, or the DNA can be physically sheared, as forexample, by sonication. The DNA fragments can then be separatedaccording to size by standard techniques, including but not limited to,agarose and polyacrylamide gel electrophoresis, column chromatographyand sucrose gradient centrifugation.

The genomic DNA or cDNA fragments can be inserted into suitable vectors,including but not limited to, plasmids, cosmids, bacteriophages lambdaor T₄, and yeast artificial chromosome (YAC) [See, for example, Sambrooket al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Glover, D.M(ed.), DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford,U.K., Vols. I and II (1985)].

The SCR nucleotide probe, DNA or RNA, should be at least 17 nucleotides,preferably at least 26 nucleotides, and most preferably at least 50nucleotides in length. The nucleotide probe is hybridized under moderatestringency conditions and washed under moderate, preferably highstringency conditions. Clones in libraries with insert DNA havingsubstantial homology to the SCR probe will hybridize to the probe.Hybridization of the nucleotide probe to genomic or cDNA libraries iscarried out using methods known in the art. One of ordinary skill in theart will know that the appropriate hybridization and wash conditionsdepend on the length and base composition of the probe and that suchconditions may be determined using standard formulae. See, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Vol. 2, (1989)pp 11.45-11.57 and 15.55-15.57.

The identity of a cloned or amplified SCR gene sequence can be verifiedby comparing the amino acid sequences of its three open reading frameswith the amino acid sequence of a SCR gene (e.g., Arabidopsis SCRprotein [SEQ ID No:2]). A SCR gene or coding sequence encodes a proteinor polypeptide whose amino acid sequence is substantially similar tothat of a SCR protein or polypeptide (e.g., the amino acid sequence ofany one of the SCR proteins and/or polypeptides shown in FIG. 5A, 5E,FIG. 8, FIG. 9, FIGS. 11A-B, FIGS. 15A-S, FIG. 17B and FIG. 18). Theidentity of the cloned or amplified SCR gene sequence may be furtherverified by examining its expression pattern, which should show highlylocalized expression in the embryo and/or root of the plant from whichthe SCR gene sequence was isolated.

Comparison of the amino acid sequences encoded by a cloned or amplifiedsequence may reveal that it does not contain the entire SCR gene or itspromoter. In such a case the cloned or amplified SCR gene sequence maybe used as a probe to screen a genomic library for clones having insertsthat overlap the cloned or amplified SCR gene sequence. A complete SCRgene and its promoter may be reconstructed by splicing the overlappingSCR gene sequences.

5.1.2. Expression of SCR Gene Products

SCR proteins, polypeptides and peptide fragments, mutated, truncated ordeleted forms of SCR and/or SCR fusion proteins can be prepared for avariety of uses, including but not limited to the generation ofantibodies, as reagents in assays, the identification of other cellulargene products involved in regulation of root development; etc.

SCR translational products include, but are not limited to thoseproteins and polypeptides encoded by the SCR gene sequences described inSection 5.1, above. The invention encompasses proteins that arefunctionally equivalent to the SCR gene products described in Section5.1. Such a SCR gene product may contain one or more deletions,additions or substitutions of SCR amino acid residues within the aminoacid sequence encoded by any one of the SCR gene sequences described,above, in Section 5.1, but which result in a silent change, thusproducing a functionally equivalent SCR gene product. Amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved.

For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Functionally equivalent”, as utilized herein, refers toa protein capable of exhibiting a substantially similar in vivo activityas the endogenous SCR gene products encoded by the SCR gene sequencesdescribed in Section 5.1, above. Alternatively, “functionallyequivalent” may refer to peptides capable of regulating gene expressionin a manner substantially similar to the way in which the correspondingportion of the endogenous SCR gene product would.

The invention also encompasses mutant SCR proteins and polypeptides thatagree not functionally equivalent to the gene products described inSection 5.1. Such a mutant SCR protein or polypeptide may contain one ormore deletions, additions or substitutions of SCR amino acid residueswithin the amino acid sequence encoded by any one the SCR gene sequencesdescribed above in Section 5.1., and which result in loss of one or morefunctions of the SCR protein (e.g., recognition of a specific nucleicsequence, binding of an transcription factor, etc.), thus producing aSCR gene product not functionally equivalent to the wild-type SCRprotein.

While random mutations can be made to SCR DNA (using random mutagenesistechniques well known to those skilled in the art) and the resultingmutant SCRs tested for activity, site-directed mutations of the SCR geneand/or coding sequence can be engineered (using site-directedmutagenesis techniques well known to those skilled in the art) togenerate mutant SCRs with increased function, (e.g., resulting inimproved root formation), or decreased function (e.g., resulting insuboptimal root function). In particular, mutated SCR proteins in whichany of the domains shown in FIGS. 13A-F are deleted or mutated arewithin the scope of the invention. Additionally, peptides correspondingto one or more domains of the SCR (e.g., shown in FIGS. 13A-F),truncated or deleted SCRs, as well as fusion proteins in which the fulllength SCR, a SCR polypeptide or peptide fused to an unrelated proteinare also within the scope of the invention and can be designed on thebasis of the SCR nucleotide and SCR amino acid sequences disclosed inSection 5.1. above.

While the SCR polypeptides and peptides can be chemically synthesized(e.g., see Creighton, 1983, Proteins: Structures and MolecularPrinciples, W.H. Freeman & Co., N.Y.) large polypeptides derived fromSCR and the full length SCR may advantageously be produced byrecombinant DNA technology using techniques well known to those skilledin the art for expressing nucleic acid sequences.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing SCR protein coding sequences andappropriate transcriptional/translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. See, forexample, the techniques described in Sambrook et al., 1989, supra, andAusubel et al., 1989, supra. Alternatively, RNA capable of encoding SCRprotein sequences may be chemically synthesized using, for example,synthesizers. See, for example, the techniques described in“Oligonucleotide Synthesis”, 1984, Gait, M. J. ed., IRL Press, Oxford.

A variety of host-expression vector systems may be utilized to expressthe SCR gene products of the invention. Such host-expression systemsrepresent vehicles by which the SCR gene products of interest may beproduced and subsequently recovered and/or purified from the culture orplant (using purification methods well known to those skilled in theart), but also represent cells which may, when transformed ortransfected with the appropriate nucleotide coding sequences, exhibitthe SCR protein of the invention in situ. These include but are notlimited to microorganisms such as bacteria (e.g., E. coli, B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing SCR protein coding sequences; yeast(e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing the SCR protein coding sequences; insectcell systems infected with recombinant virus expression vectors (e.g.,baculovirus) containing the SCR protein coding sequences; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing SCR protein coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter; thecytomegalovirus promoter/enhancer;

etc.).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the SCRprotein being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of antibodies or to screenpeptide libraries, for example, vectors which direct the expression ofhigh levels of fusion protein products that are readily purified may bedesirable. Such vectors include, but are not limited, to the E. coliexpression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in whichthe SCR coding sequence may be ligated individually into the vector inframe with the lac Z coding region so that a fusion protein is produced;pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.pGEX vectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene protein can be released from the GST moiety.

In one such embodiment of a bacterial system, full length cDNA sequencesare appended with in-frame Bam HI sites at the amino terminus and Eco RIsites at the carboxyl terminus using standard PCR methodologies (Inniset al., 1990, supra) and ligated into the pGEX-2TK vector (Pharmacia,Uppsala, Sweden). The resulting cDNA construct contains a kinaserecognition site at the amino terminus for radioactive labelling andglutathione S-transferase sequences at the carboxyl terminus foraffinity purification (Nilsson, et al., 1985, EMBO J. 4: 1075; Zabeauand Stanley, 1982, EMBO J. 1: 1217.

The recombinant constructs of the present invention may include aselectable marker for propagation of the construct. For example, aconstruct to be propagated in bacteria preferably contains an antibioticresistance gene, such as one that confers resistance to kanamycin,tetracycline, streptomycin, or chloramphenicol. Suitable vectors forpropagating the construct include plasmids, cosmids, bacteriophages orviruses, to name but a few.

In addition, the recombinant constructs may include plant-expressible,selectable, or screenable marker genes for isolating, identifying ortracking plant cells transformed by these constructs. Selectable markersinclude, but are not limited to, genes that confer antibioticresistance, (e.g., resistance to kanamycin or hygromycin) or herbicideresistance (e.g., resistance to sulfonylurea, phosphinothricin, orglyphosate). Screenable markers include, but are not be limited to,genes encoding β-glucuronidase (Jefferson, 1987, Plant Mol. Biol. Rep.5:387-405), luciferase (Ow et al., 1986, Science 234:856-859), B proteinthat regulates anthocyanin pigment production (Goff et al., 1990, EMBO J9:2517-2522).

In embodiments of the present invention which utilize the Agrobacteriumtumefacien system for transforming plants (see infra), the recombinantconstructs may additionally comprise at least the right T-DNA bordersequences flanking the DNA sequences to be transformed into the plantcell. Alternatively, the recombinant constructs may comprise the rightand left T-DNA border sequences flanking the DNA sequence. The properdesign and construction of such T-DNA based transformation vectors arewell known to those skilled in the art.

5.1.3. Antibodies to SCR Proteins and Polypeptides

Antibodies that specifically recognize one or more epitopes of SCR, orepitopes of conserved variants of SCR, or peptide fragments of the SCRare also encompassed by the invention. Such antibodies include but arenot limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

For the production of antibodies, various host animals may be immunizedby injection with the SCR protein, an SCR peptide (e.g., onecorresponding to a functional domain of the protein), a truncated SCRpolypeptide (SCR in which one or more domains has been deleted),functional equivalents of the SCR protein, or mutants of the SCRprotein. Such SCR proteins, polypeptides, peptides or fusion proteinscan be prepared and obtained as described in Section 5.1.2. supra. Hostanimals may include but are not limited to rabbits, mice, and rats, toname but a few. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (Nature 256:495-497 [1975]; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

In addition, techniques have been developed for the production ofhumanized antibodies. (See, e.g., Queen, U.S. Pat. No. 5,585,089.) Animmunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions, referredto as complementarily determining regions (CDRs). The extent of theframework region and CDRs have been precisely defined (see, “Sequencesof Proteins of Immunological Interest”, Kabat, E. et al., U.S.Department of Health and Human Services (1983). Briefly, humanizedantibodies are antibody molecules from non-human species having one ormore CDRs from the non-human species and a framework region from a humanimmunoglobulin molecule.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to produce single chainantibodies against SCR proteins or polypeptides. Single chain antibodiesare formed by linking the heavy and light chain fragments of the Fvregion via an amino acid bridge, resulting in a single chainpolypeptide.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies to a SCR protein and/or polypeptide can, in turn, be utilizedto generate anti-idiotype antibodies that “mimic” SCR, using techniqueswell known to those skilled in the art. (See, e.g., Greenspan & Bona,1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438).

5.1.4. SCR Gene or Gene Products as Markers for Qualitative Trait Loci

Any of the nucleotide sequences (including EST clone sequences)described in §§5.1 and 5.1.1. and/or listed in Table 1, and/orpolypeptides and proteins described in §§5.1.2. and/or listed in Table1, can be used as markers for qualitative trait loci in breedingprograms for crop plants. To this end, the nucleic acid molecules,including but not limited to full length SCR coding sequences, and/orpartial sequences (ESTs), can be used in hybridization and/or DNAamplification assays to identify the endogenous SCR genes, scr mutantalleles and/or SCR expression products in cultivars as compared towild-type plants. They can also be used as markers for linkage analysisof qualitative trait loci. It is also possible that the SCR gene mayencode a product responsible for a qualitative trait that is desirablein a crop breeding program. Alternatively, the SCR protein, peptidesand/or antibodies can be used as reagents in immunoassays to detectexpression of the SCR gene in cultivars and wild-type plants.

5.2. SCR Promoters

According to the present invention, SCR promoters and functionalportions thereof described herein refer to regions of the SCR gene whichare capable of promoting tissue-specific expression in embryos and/orroots of an operably linked coding sequence in plants. The SCR promoterdescribed herein refers to the regulatory elements of SCR genes, i.e.,regulatory regions of genes which are capable of selectively hybridizingto the nucleic acids described in Section 5.1, or regulatory sequencescontained, for example, in the region between the translational startsite of the Arabidopsis SCR gene and the HindIII site approximately 2.5kb upstream of the site in plasmid pLIG1-3/SAC+Mob21SAC (see FIGS. 5Aand 14) in hybridization assays, or which are homologous by sequenceanalysis (containing a span of 10 or more nucleotides in which at least50 percent of the nucleotides are identical to the sequences presentedherein). Homologous nucleotide sequences refer to nucleotide sequencesincluding, but not limited to, SCR promoters in diverse plant species(e.g., promoters of orthologs of Arabidopsis SCR) as well as geneticallyengineered derivatives of the promoters described herein.

Methods which could be used for the synthesis, isolation, molecularcloning, characterization and manipulation of SCR promoter sequences arewell known to those skilled in the art. See, e.g., the techniquesdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd. ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1989).

According to the present invention, SCR promoter sequences or portionsthereof described herein may be obtained from appropriate plant ormammalian sources from cell lines or recombinant DNA constructscontaining SCR promoter sequences, and/or by chemical synthetic methods.SCR promoter sequences can be obtained from genomic clones containingsequences 5′ upstream of SCR coding sequences. Such 5′ upstream clonesmay be obtained by screening genomic libraries using SCR protein codingsequences, particularly those encoding SCR N-terminal sequences, fromSCR gene clones obtained as described in Sections 5.1. and 5.2. Standardmethods that may used in such screening include, for example, the methodset forth in Benton & Davis, 1977, Science 196:180 for bacteriophagelibraries; and Grunstein & Hogness, 1975, Proc. Nat. Acad. Sci. U.S.A.72:3961-3965 for plasmid libraries.

The full extent and location of SCR promoters within such 5′ upstreamclones may be determined by the functional assay described below. In theevent a 5′ upstream clone does not contain the entire SCR promoter asdetermined by the functional assay, the insert DNA of the clone may beused to isolate genomic clones containing sequences further 5′ upstreamof the SCR coding sequences. Such further upstream sequences can bespliced on to existing 5′ upstream sequences and the reconstructed 5′upstream region tested for functionality as a SCR promoter (i.e.,promoting tissue-specific expression in embryos and/or roots of anoperably linked gene in plants). This process may be repeat until thecomplete SCR promoter is obtained.

The location of the SCR promoter within genomic sequences 5′ upstream ofthe SCR gene isolated as described above may be determined using anymethod known in the art. For example, the 3′-end of the promoter may beidentified by locating the transcription initiation site, which may bedetermined by methods such as RNase protection (e.g., Liang et al.,1989, J. Biol. Chem. 264:14486-14498), primer extension (e.g.,Weissenborn & Larson, 1992, J. Biol. Chem. 267:6122-6131), and/orreverse transcriptase/PCR. The location of the 3′-end of the promotermay be confirmed by sequencing and computer analysis, examining for thecanonical AGGA or TATA boxes of promoters that are typically 50-60 basepairs (bp) and 25-35 bp 5′-upstream of the transcription initiationsite. The 5′-end promoter may be defined by deleting sequences from the5′-end of the promoter containing fragment, constructing atranscriptional or translational fusion of the resected fragment and areporter gene, and examining the expression characteristics of thechimeric gene in transgenic plants. Reporter genes that may be used tosuch ends include, but are not limited to, GUS, CAT, luciferase,β-galactosidase and C1 and R gene controlling anthocyanin production.

According to the present invention, a SCR promoter is one that confersto an operably linked gene in a transgenic plant tissue-specificexpression in roots, root nodules, stems and/or embryos. A SCR promotercomprises the region between about −5,000 bp and +1 bp upstream of thetranscription initiation site of SCR gene. In a particular embodiment,the Arabidopsis SCR promoter comprises the region between positions −2.5kb and +1 in the 5′ upstream region of the Arabidopsis SCR gene (seeFIGS. 5A and 14).

5.2.1. Cis-Regulatory Elements of SCR Promoters

According to the present invention, the cis-regulatory elements within aSCR promoter may be identified using any method known in the art. Forexample, the location of cis-regulatory elements within an induciblepromoter may be identified using methods such as DNase or chemicalfootprinting (e.g., Meier et al., 1991, Plant Cell 3:309-315) or gelretardation (e.g., Weissenborn & Larson, 1992, J. Biol. Chem.267-6122-6131; Beato, 1989, Cell 56:335-344; Johnson et al., 1989, Ann.Rev. Biochem. 58:799-839). Additionally, resectioning experiments mayalso be employed to define the location of the cis-regulatory elements.For example, an inducible promoter-containing fragment may be resectedfrom either the 5′ or 3′-end using restriction enzyme or exonucleasedigests.

To determine the location of cis-regulatory elements within the sequencecontaining the inducible promoter, the 5′- or 3′-resected fragments,internal fragments to the inducible promoter containing sequence, orinducible promoter fragments containing sequences identified byfootprinting or gel retardation experiments may be fused to the 5′-endof a truncated plant promoter, and the activity of the chimeric promoterin transgenic plant examined. Useful truncated promoters to these endscomprise sequences starting at or about the transcription initiationsite and extending to no more than 150 bp 5′ upstream. These truncatedpromoters generally are inactive or are only minimally active. Examplesof such truncated plant promoters may include, among others, a “minimal”CaMV 35S promoter whose 5′ end terminates at position −46 bp withrespect to the transcription initiation site (Skriver et al., Proc.Natl. Acad. Sci. USA 88:7266-7270); the truncated “−90 35S” promoter inthe X-GUS-90 vector (Benfey & Chua, 1989, Science 244:174-181); atruncated “−101 nos” promoter derived from the nopaline synthasepromoter (Aryan et al., 1991, Mol. Gen. Genet. 225:65-71); and thetruncated maize Adh-1 promoter in pADcat 2 (Ellis et al., 1987, EMBO J.6:11-16).

According to the present invention, a cis-regulatory element of a SCRpromoter is a sequence that confers to a truncated promotertissue-specific expression in embryos, stems, root nodules and/or roots.

5.2.2. SCR Promoter-Driven Expression Vectors

The properties of the nucleic acid sequences are varied as are thegenetic structures of various potential host plant cells. In thepreferred embodiments of the present invention, described herein, anumber of features which an artisan may recognize as not beingabsolutely essential, but clearly advantageous are used. These includemethods of isolation, synthesis or construction of gene constructs, themanipulation of the gene constructs to be introduced into plant cells,certain features of the gene constructs, and certain features of thevectors associated with the gene constructs.

Further, the gene constructs of the present invention may be encoded onDNA or RNA molecules. According to the present invention, it ispreferred that the desired, stable genotypic change of the target plantbe effected through genomic integration of exogenously introducednucleic acid construct(s), particularly recombinant DNA constructs.Nonetheless, according to the present invention, such genotypic changescan also be effected by the introduction of episomes (DNA or RNA) thatcan replicate autonomously and that are somatically and germinallystable. Where the introduced nucleic acid constructs comprise RNA, planttransformation or gene expression from such constructs may proceedthrough a DNA intermediate produced by reverse transcription.

The present invention provides for use of recombinant DNA constructswhich contain tissue-specific and developmental-specific promoterfragments and functional portions thereof. As used herein, a functionalportion of a SCR promoter is capable of functioning as a tissue-specificpromoter in the embryo, stem, root nodule and/or root of a plant. Thefunctionality of such sequences can be readily established by any methodknown in the art. Such methods include, for example, constructingexpression vectors with such sequences and determining whether theyconfer tissue-specific expression in the embryo, stem, root noduleand/or root to an operably linked gene. In a particular embodiment, theinvention provides for the use of the Arabidopsis SCR promoter containedin the sequences depicted in FIGS. 5A and 14 and the insert DNA ofplasmid pGEX-2TK⁺.

The SCR promoters of the invention may be used to direct the expressionof any desired protein, or to direct the expression of a RNA product,including, but not limited to, an “antisense” RNA or ribozyme. Suchrecombinant constructs generally comprise a native SCR promoter or arecombinant SCR promoter derived therefrom, ligated to the nucleic acidsequence encoding a desired heterologous gene product.

A recombinant SCR promoter is used herein to refer to a promoter thatcomprises a functional portion of a native SCR promoter or a promoterthat contains native promoter sequences that is modified by a regulatoryelement from a SCR promoter. Alternatively, a recombinant induciblepromoter derived from the scr promoter may be a chimeric promoter,comprising a full-length or truncated plant promoter modified by theattachment of one or more SCR cis-regulatory elements.

The manner of chimeric promoter constructions may be any well known inthe art. For examples of approaches that can be used in suchconstructions, see Section 5.1.2., above and Fluhr et al., 1986, Science232:1106-1112; Ellis et al., 1987, EMBO J. 6:11-16; Strittmatter & Chua,1987, Proc. Natl. Acad. Sci. USA 84:8986-8990; Poulsen & Chua, 1988,Mol. Gen. Genet. 214:16-23; Comai et al., 1991, Plant Mol. Biol.15:373-381; Aryan et al., 1991, Mol. Gen. Genet. 225:65-71.

According to the present invention, where a SCR promoter or arecombinant SCR promoter is used to express a desired protein, the DNAconstruct is designed so that the protein coding sequence is ligated inphase with the translational initiation codon downstream of thepromoter. Where the promoter fragment is missing 5′ leader sequences, aDNA fragment encoding both the protein and its 5′ RNA leader sequence isligated immediately downstream of the transcription initiation site.Alternatively, an unrelated 5′ RNA leader sequence may be used to bridgethe promoter and the protein coding sequence. In such instances, thedesign should be such that the protein coding sequence is ligated inphase with the initiation codon present in the leader sequence, orligated such that no initiation codon is interposed between thetranscription initiation site and the first methionine codon of theprotein.

Further, it may be desirable to include additional DNA sequences in theprotein expression constructs. Examples of additional DNA sequencesinclude, but are not limited to, those encoding: a 3′ untranslatedregion; a transcription termination and polyadenylation signal; anintron; a signal peptide (which facilitates the secretion of theprotein); or a transit peptide (which targets the protein to aparticular cellular compartment such as the nucleus, chloroplast,mitochondria, or vacuole).

5.3. Production of Transgenic Plants and Plant Cells

According to the present invention, a desirable plant or plant cell maybe obtained by transforming a plant cell with the nucleic acidconstructs described herein. In some instances, it may be desirable toengineer a plant or plant cell with several different gene constructs.Such engineering may be accomplished by transforming a plant or plantcell with all of the desired gene constructs simultaneously.Alternatively, the engineering may be carried out sequentially. That is,transforming with one gene construct, obtaining the desired transformantafter selection and screening, transforming the transformant with asecond gene construct, and so on.

In an embodiment of the present invention, Agrobacterium is employed tointroduce the gene constructs into plants. Such transformationspreferably use binary Agrobacterium T-DNA vectors (Bevan, 1984, Nuc.Acid Res. 12:8711-8721), and the co-cultivation procedure (Horsch etal., 1985, Science 227:1229-1231). Generally, the Agrobacteriumtransformation system is used to engineer dicotyledonous plants (Bevanet al., 1982, Ann. Rev. Genet. 16:357-384; Rogers et al., 1986, MethodsEnzymol. 118:627-641). The Agrobacterium transformation system may alsobe used to transform, as well as transfer, DNA to monocotyledonousplants and plant cells (see Hernalsteen et al., 1984, EMBO J3:3039-3041; Hooykass-Van Slogteren et al., 1984, Nature 311:763-764;Grimsley et al., 1987, Nature 325:1677-179; Boulton et al., 1989, PlantMol. Biol. 12:31-40.; Gould et al., 1991, Plant Physiol. 95:426-434).

In other embodiments, various alternative methods for introducingrecombinant nucleic acid constructs into plants and plant cells may alsobe utilized. These other methods are particularly useful where thetarget is a monocotyledonous plant or plant cell. Alternative genetransfer and transformation methods include, but are not limited to,protoplast transformation through calcium-, polyethylene glycol (PEG)-or electroporation-mediated uptake of naked DNA (see Paszkowski et al.,1984, EMBO J 3:2717-2722, Potrykus et al., 1985, Mol. Gen. Genet.199:169-177; Fromm et al., 1985, Proc. Natl. Acad. Sci. USA82:5824-5828; Shimamoto, 1989, Nature 338:274-276), and electroporationof plant tissues (D'Halluin et al., 1992, Plant Cell 4:1495-1505).Additional methods for plant cell transformation include microinjection,silicon carbide mediated DNA uptake (Kaeppler et al., 1990, Plant CellReporter 9:415-418), and microprojectile bombardment (see Klein et al.,1988, Proc. Natl. Acad. Sci. USA 85:4305-4309; Gordon-Kamm et al., 1990,Plant Cell 2:603-618).

According to the present invention, a wide variety of plants may beengineered for the desired physiological and agronomic characteristicsdescribed herein using the nucleic acid constructs of the instantinvention and the various transformation methods mentioned above. Inpreferred embodiments, target plants for engineering include, but arenot limited to, crop plants such as maize, wheat, rice, soybean, tomato,tobacco, carrots, peanut, potato, sugar beets, sunflower, yam,Arabidopsis, rape seed, and petunia; and trees such as spruce.

According to the present invention, desired plants and plant cells maybe obtained by engineering the gene constructs described herein into avariety of plant cell types, including but not limited to, protoplasts,tissue culture cells, tissue and organ explants, pollen, embryos as wellas whole plants. In an embodiment of the present invention, theengineered plant material is selected or screened for transformants(i.e., those that have incorporated or integrated the introduced geneconstruct(s)) following the approaches and methods described below. Anisolated transformant may then be regenerated into a plant.Alternatively, the engineered plant material may be regenerated into aplant, or plantlet, before subjecting the derived plant, or plantlet, toselection or screening for the marker gene traits. Procedures forregenerating plants from plant cells, tissues or organs, either beforeor after selecting or screening for marker gene(s), are well known tothose skilled in the art.

A transformed plant cell, callus, tissue or plant may be identified andisolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the transforming DNA. Forinstance, selection may be performed by growing the engineered plantmaterial on media containing inhibitory amounts of the antibiotic orherbicide to which the transforming marker gene construct confersresistance. Further, transformed plants and plant cells may also beidentified by screening for the activities of any visible marker genes(e.g., the β-glucuronidase, luciferase, B or C1 genes) that may bepresent on the recombinant nucleic acid constructs of the presentinvention. Such selection and screening methodologies are well known tothose skilled in the art.

Physical and biochemical methods may also be used to identify a plant orplant cell transformant containing the gene constructs of the presentinvention. These methods include but are not limited to: 1) Southernanalysis or PCR amplification for detecting and determining thestructure of the recombinant DNA insert; 2) Northern blot, S-1 RNaseprotection, primer-extension or reverse transcriptase-PCR amplificationfor detecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct; 4) protein gelelectrophoresis, western blot techniques, immunoprecipitation, orenzyme-linked immunoassays, where the gene construct products areproteins; 5) biochemical measurements of compounds produced as aconsequence of the expression of the introduced gene constructs.Additional techniques, such as in situ hybridization, enzyme staining,and immunostaining, may also be used to detect the presence orexpression of the recombinant construct in specific plant organs andtissues. The methods for doing all these assays are well known to thoseskilled in the art.

5.3.1. Transgenic Plants that Ectopically Express SCR

In accordance to the present invention, a plant that expresses arecombinant SCR gene may be engineered by transforming a plant cell witha gene construct comprising a plant promoter operably associated with asequence encoding SCR protein or a fragment thereof. (Operablyassociated is used herein to mean that transcription controlled by the“associated” promoter would produce a functional messenger RNA, whosetranslation would produce the enzyme.) The plant promoter may beconstitutive or inducible. Useful constitutive promoters include, butare not limited to, the CaMV 35S promoter, the T-DNA mannopinesynthetase promoter, and their various derivatives. Useful induciblepromoters include but are not limited to the promoters of ribulosebisphosphate carboxylase (RUBISCO) genes, chlorophyll a/b bindingprotein (CAB) genes, heat shock genes, the defense responsive gene(e.g., phenylalanine ammonia lyase genes), wound induced genes (e.g.,hydroxyproline rich cell wall protein genes), chemically-inducible genes(e.g., nitrate reductase genes, gluconase genes, chitinase genes, PR-1genes etc.), dark-inducible genes (e.g., asparagine synthetase gene(Coruzzi and Tsai, U.S. Pat. No. 5,256,558, Oct. 26, 1993, Gene EncodingPlant Asparagine Synthetase) developmentally regulated genes (e.g.,Shoot Meristemless gene) to name just a few.

In yet another embodiment of the present invention, it may beadvantageous to transform a plant with a gene construct operably linkinga modified or artificial promoter to a sequence encoding SCR protein ora fragment thereof. Typically, such promoters, constructed byrecombining structural elements of different promoters, have uniqueexpression patterns and/or levels not found in natural promoters. See,e.g., Salina et al., 1992, Plant Cell 4:1485-1493, for examples ofartificial promoters constructed from combining cis-regulatory elementswith a promoter core.

In a preferred embodiment of the present invention, the associatedpromoter is a strong and root, root nodule, stem and/or embryo-specificplant promoter such that the SCR protein is overexpressed in thetransgenic plant. Examples of root- and root nodules-specific promotersinclude but are not limited to the promoters of SCR genes, SHR genes,legehemoglobin genes, nodulin genes and root-specific glutaminesynthetase genes (See e.g., Tingey et al., 1987, EMBO J. 6:1-9; Edwardset al., 1990, Proc. Nat. Acad. Sci. USA 87:3459-3463).

In yet another preferred embodiment of the present invention, theoverexpression of SCR protein in roots may be engineered by increasingthe copy number of the SCR gene. One approach to producing suchtransgenic plants is to transform with nucleic acid constructs thatcontain multiple copies of the complete SCR gene (i.e., with its ownnative scr promoter). Another approach is repeatedly transformsuccessive generations of a plant line with one or more copies of thecomplete SCR gene. Yet another approach is to place a complete SCR genein a nucleic acid construct containing an amplification-selectablemarker (ASM) gene such as the glutamine synthetase or dihydrofolatereductase gene. Cells transformed with such constructs is subjected toculturing regimes that select cell lines with increased copies ofcomplete SCR gene. See, e.g., Donn et al., 1984, J. Mol. Appl. Genet.2:549-562, for a selection protocol used to isolate of a plant cell linecontaining amplified copies of the GS gene. Because the desired gene isclosely linked to the ASM, cell lines that amplified the ASM gene arealso likely to have amplified the SCR gene. Cell lines with amplifiedcopies of the SCR gene can then be regenerated into transgenic plants.

5.3.2. Transgenic Plants that Suppress Endogenous SCR Expression

In accordance with the present invention, a desired plant may beengineered by suppressing SCR activity. In one embodiment, thesuppression may be engineered by transforming a plant with a geneconstruct encoding an antisense RNA or ribozyme complementary to asegment or the whole of SCR RNA transcript, including the mature targetmRNA. In another embodiment, SCR gene suppression may be engineered bytransforming a plant cell with a gene construct encoding a ribozyme thatcleaves the SCR mRNA transcript. Alternatively, the plant can beengineered, e.g., via targeted homologous recombination to inactive or“knock-out” expression of the plant's endogenous SCR.

For all of the aforementioned suppression constructs, it is preferredthat such gene constructs express specifically in the root, root nodule,stem and/or embryo tissues. Alternatively, it may be preferred to havethe suppression constructs expressed constitutively. Thus, constitutivepromoters, such as the nopaline, CaMV 35S promoter, may also be used toexpress the suppression constructs. A most preferred promoter for thesesuppression constructs is a SCR or SHR promoter.

In accordance with the present invention, desired plants with suppressedtarget gene expression may also be engineered by transforming a plantcell with a co-suppression construct. A co-suppression constructcomprises a functional promoter operatively associated with a completeor partial SCR gene sequence. It is preferred that the operativelyassociated promoter be a strong, constitutive promoter, such as the CaMV35S promoter. Alternatively, the co-suppression construct promoter canbe one that expresses with the same tissue and developmental specificityas the scr gene.

According to the present invention, it is preferred that theco-suppression construct encodes a incomplete SCR mRNA, although aconstruct encoding a fully functional SCR mRNA or enzyme may also beuseful in effecting co-suppression.

In accordance with the present invention, desired plants with suppressedtarget gene expression may also be engineered by transforming a plantcell with a construct that can effect site-directed mutagenesis of theSCR gene. (See, e.g., Offringa et al., 1990, EMBO J. 9:3077-84; andKanevskii et al., 1990, Dokl. Akad. Nauk. SSSR 312:1505-1507) fordiscussions of nucleic constructs for effecting site-directedmutagenesis of target genes in plants.) It is preferred that suchconstructs effect suppression of SCR gene by replacing the endogenousSCR gene sequence through homologous recombination with none or inactiveSCR protein coding sequence.

5.3.3. Transgenic Plants that Express a Transgene Controlled by the SCRPromoter

In accordance with the present invention, a desired plant may beengineered to express a gene of interest under the control of the SCRpromoter. SCR promoters and functional portions thereof refer to regionsof the nucleic acid sequence which are capable of promotingtissue-specific transcription of an operably linked gene of interest inthe embryo, stem, root nodule and/or root of a plant. The SCR promoterdescribed herein refers to the regulatory elements of SCR genes asdescribed in Section 5.2.

Genes that may be beneficially expressed in the roots and/or rootnodules of plants include genes involved in nitrogen fixation orcytokines or auxins, or genes which regulate growth, or growth of roots.In addition, genes encoding proteins that confer on plants herbicide,salt, or pest resistance may be engineered for root specific expression.The nutritional value of root crops may also be enhanced through SCRpromoter driven expression of nutritional proteins. Alternatively,therapeutically useful proteins may be expressed specifically in rootcrops.

Genes that may be beneficially expressed in the stems of plants includethose involved in starch lignin or cellulose biosynthesis.

In accordance with the present invention, desired plants which express aheterologous gene of interest under the control of the SCR promoter maybe engineered by transforming a plant cell with SCR promoter drivenconstructs using those techniques described in Section 5.2.2. and 5.3.,supra.

5.3.4. Screening of Transformed Plants for Those Having Desired AlteredTraits

It will be recognized by those skilled in the art that in order toobtain transgenic plants having the desired engineered traits, screeningof transformed plants (i.e., those having an gene construct of theinvention) having those traits may be required. For example, where theplants have been engineered for ectopic overexpression of SCR gene,transformed plants are examined for those expressing the SCR gene at thedesired level and in the desired tissues and developmental stages. Wherethe plants have been engineered for suppression of the SCR gene product,transformed plants are examined for those expressing the SCR geneproduct (e.g., RNA or protein) at reduced levels in various tissues. Theplants exhibiting the desired physiological changes, e.g., ectopic SCRoverexpression or SCR suppression, may then be subsequently screened forthose plants that have the desired structural changes at the plant level(e.g., transgenic plants with overexpression or suppression of SCR genehaving the desired altered root structure). The same principle appliesto obtaining transgenic plants having tissue-specific expression of aheterologous gene in embryos and/or roots by the use of a SCR promoterdriven expression construct.

Alternatively, the transformed plants may be directly screened for thoseexhibiting the desired structural and functional changes. In oneembodiment, such screening may be for the size, length or pattern of theroot of the transformed plants. In another embodiment, the screening ofthe transformed plants may be for altered gravitropism or decreasedsusceptibility to lodging. In other embodiments, the screening of thetransformed plants may be for improved agronomic characteristics (e.g.,faster growth, greater vegetative or reproductive yields, or improvedprotein contents, etc.), as compared to unengineered progenitor plants,when cultivated under various growth conditions (e.g., soils or mediacontaining different amount of nutrients, water content).

According to the present invention, plants engineered with SCRoverexpression may exhibit improved vigorous growth characteristics whencultivated under conditions where large and thicker roots areadvantageous. Plants engineered for SCR suppression may exhibit improvedvigorous growth characteristics when cultivated under conditions wherethinner roots are advantageous.

Engineered plants and plant lines possessing such improved agronomiccharacteristics may be identified by examining any of followingparameters: 1) the rate of growth, measured in terms of rate of increasein fresh or dry weight; 2) vegetative yield of the mature plant, interms of fresh or dry weight; 3) the seed or fruit yield; 4) the seed orfruit weight; 5) the total nitrogen content of the plant; 6) the totalnitrogen content of the fruit or seed; 7) the free amino acid content ofthe plant; 8) the free amino acid content of the fruit or seed; 9) thetotal protein content of the plant; and 10) the total protein content ofthe fruit or seed. The procedures and methods for examining theseparameters are well known to those skilled in the art.

According to the present invention, a desired plant is one that exhibitsimprovement over the control plant (i.e., progenitor plant) in one ormore of the aforementioned parameters. In an embodiment, a desired plantis one that shows at least 5% increase over the control plant in atleast one parameter. In a preferred embodiment, a desired plant is onethat shows at least 20% increase over the control plant in at least oneparameter. Most preferred is a plant that shows at least 50% increase inat least one parameter.

EXAMPLE 1: Arabidopsis SCR Gene

This example describes the cloning and structure of the Arabidopsis SCRgene and its expression. The deduced amino acid sequence of theArabidopsis SCR gene product contains a number of potential functionaldomains similar to those found in transcription factors. Closely relatedsequences have been found in both dicots and monocots indicating thatArabidopsis SCR is a member of a new protein family. The expressionpattern of the SCR gene was characterized by means of in situhybridization and by an enhancer trap insertion upstream of the SCR gene(described in more detail in Section 7). The expression pattern isconsistent with a key role for Arabidopsis SCR in regulating theasymmetric division of the cortex/endodermis initial which is essentialfor generating the radial organization of the root.

6.1. Materials and Methods

6.1.1. Plant Culture

Arabidopsis ecotypes Wassilewskija (Ws), Columbia (Col), and Landsbergerecta (Ler) were obtained from Lehle. Arabidopsis seeds were surfacesterilized and grown as described previously (Benfey et al., 1993,Development 119:57-70). Generation of the enhancer trap lines isdescribed in Section 7.

6.1.2. Genetic Analysis

For the scr-1 allele, co-segregation of the mutant phenotype andkanamycin resistance conferred by the inserted T-DNA was determined asdescribed previously (Aeschbacher et al., 1995, Genes & Development9:330-340). Because kanamycin affects root growth, 1557 seeds fromheterozygous lines were germinated on non-selective media, scored forthe appearance of the mutant phenotype, and subsequently transferred toselective media. All (284) phenotypically mutant seedlings showedresistance to the antibiotic, whereas 834 of 1273 phenotypicallywild-type seedlings showed resistance to kanamycin, respectively.Phenotypically wild type plants (83) were also transferred to soil andallowed to set seeds. The progeny of these plants were plated onselective and non-selective media, and scored for the co-segregation ofthe mutant phenotype and antibiotic resistance. A majority (48) of theplants segregated for the mutant phenotype and for kanamycin resistance,whereas 35 were wild-type and sensitive to kanamycin. Due to amis-identified cross, scr-2 was originally thought to be non-allelic andwas named pinocchio (Scheres et al., 1995, Development 121:53-62).Subsequent mapping results placed it in an identical chromosomallocation as scr-1. The original scr-2 line contained at least two T-DNAinserts. Co-segregation analysis revealed a lack of linkage between theantibiotic resistance marker carried by the T-DNA and the mutantphenotype. Antibiotic sensitive lines were identified that segregatedfor mutants. These lines were crossed to scr-1. All F1 antibioticresistant progeny exhibited a mutant phenotype. All F2 progeny (fromindependent lines) were mutant, and there was a 3:1 segregation forantibiotic resistance indicating that the two mutations were allelic.Antibiotic sensitive lines of scr-2 were found to contain a rearrangedT-DNA insert as determined by Southern blots and PCR using T-DNAspecific probes and primers respectively. The presence of this T-DNA inthe SCR gene was confirmed by Southern blots using SCR probes. Acombination of T-DNA and SCR specific primers was used to amplifyT-DNA/SCR junctions. The PCR fragments were cloned using the TA cloningkit (Invitrogen) and sequenced. The insertion points were determined forboth 5′ and 3′ T-DNA/SCR junctions.

6.1.3. MAPPING

Mutant plants of scr-2 (WS background) were crossed to Col WT. DNA frommutant F2 individual plants were analyzed for co-segregation withmicrosatellite (Bell & Ecker, 1994, Genomics 18:137-144) and CAPSmarkers (Konieczny & Ausubel, 1993, Plant J. 4:403-410). The closestlinkage was found to two CAPS markers located at the bottom ofchromosome III. Only one out of 238 mutant chromosomes was recombinantfor the BGL1 marker (Konieczny & Ausubel, 1993, Plant J. 4:403-410) andone out of 210 chromosomes was recombinant for the cdc2b marker.

A RFLP for the SCR gene was identified between Col and Ler ecotypes withXho I endonuclease. Genomic DNAs from independent R1 lines (Jarvis etal., 1994, Plant Mol. Biol. 24:685-687) were digested with Xho I andblots were hybridized to SCR. Using the segregation data obtained for R1lines, the SCR gene was mapped relative to molecular markers by CLUSTER.The SCR gene was assigned to the bottom of chromosome III closest toBGL1.

6.1.4. Phenotypic Analysis

Morphological characterization of the mutant roots was performed asfollows: 7 to 14 days post-germination phenotypically mutant seedlingswere fixed in 4.0% formaldehyde in PIPES buffer pH 7.2. After fixationthe samples were dehydrated in ethanol followed by infiltration withHistoresin (Jung-Leica, Heidelberg, Germany). Plastic sections weremounted on superfrost slides (Fisher). The sections were either stainedwith 0.05% toluidine blue and photographed using Kodak 160 T film orused for Casparian strip detection or antibody staining.

Casparian strip detection was performed as described previously (Schereset al., 1995, Development 121:53-62), with the following modifications.Plastic sections were used and the counterstaining was done in 0.1%aniline blue for 5 to 15 min. The sections were visualized with a Leitzfluorescent microscope with FITC filter. Pictures were taken using aLeitz camera attached to the microscope and Kodak HC400 film. Slideswere digitized with a Nikon slide scanner and manipulated in AdobePhotoshop.

For antibody staining, sections were blocked for 2 hours at roomtemperature in 1% BSA in PBS containing 0.1% Tween 20 (PBT). Sampleswere incubated with primary antibodies at 4° C. in 1% BSA in PBTovernight, and then washed 3 times 5 minutes each with PBT. Samples wereincubated for two hours with biotinylated secondary antibodies (VectorLaboratories) in PBT, and washed as above. Samples were incubated withTexas Red conjugated avidin D for 2 hours at room temperature, washed asbefore, and mounted in Citifluor. Immunofluorescence was observed with afluorescent microscope equipped with a Rhodamine filter. Staining withthe CCRC antibodies was performed as described previously (Freshour etal., 1996, Plant Physiol. 110:1413-1429).

6.1.5. Molecular Techniques

Genomic DNA preparation was performed using the Elu-Quik kit (Schleicher& Schuell) protocol. Radioactive and non-radioactive DNA probes werelabeled with either random primed labeling or PCR-mediated synthesisaccording to the Genius kit manual (Boehringer Mannheim). E. coli andAgrobacterium tumefaciens cells were transformed using a BIO-RAD genepulser. Plasmid DNA was purified using the alkaline lysis method(Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y.:Cold Spring Harbor Laboratory, 1982).

A probe made from a rescued fragment of 1.2 kb was used to screen awild-type genomic library made from WS plants. One genomic clonecontaining an insert of approximately 23 kb was isolated. A 3.0 kb Sac Ifragment from the genomic clone, which hybridized to the 1.2 kb probe,was subcloned and sequenced (FIG. 5A). Comparison of the nucleotidesequence between the genomic clone and the rescued plasmid revealed thesite of the T-DNA insertion. Approximately 600,000 plaques from a cDNAlibrary, obtained from inflorescences and siliques (Col ecotype), andtherefore enriched in embryos, were screened with the 1.2 kb probe. FourcDNA clones were isolated. The dideoxy sequencing method was performedusing the Sequenase kit (United States Biochemical Corp.).Sequence-specific internal primers were synthesized and used to sequencethe Sac I genomic as well the cDNA clones. Total RNA from plant tissueswas obtained using phenol/chloroform extractions as described in (Berryet al., 1985, Mol. Cell. Biol. 5:2238-2246) with minor modifications.Northern hybridization and detection were performed according to theGenius kit manual (Boehringer Mannheim).

To identify the site of insertion of the enhancer-trap T-DNA, genomicDNA from ET199 homozygous plants was amplified using primers specificfor the T-DNA left border and the SCR gene. An approximately 2.0 kbfragment was amplified. This fragment was sequenced and the site ofinsertion was found to be approximately 1 kb from the ATG start codon.

6.1.6. In Situ Hybridization

Antisense and sense SCR riboprobes were labeled with digoxigenin-11-UTP(Boehringer Mannheim) using T7 polymerase following the manufacturer'sprotocol. Probes contained a 1.1 kb 3′ portion of the cDNA. Probepurification, hydrolysis and quantification were performed as describedin the Boehringer Mannheim Genius System user's guide.

Tissue samples were fixed in 4% formaldehyde overnight at 4° C. andrinsed two times in PBS (Jackson et al., 1991, Pl. Cell 3:115-125). Theywere subsequently pre-embedded in 1% agarose in PBS. The fixed tissuewas dehydrated in ethanol, cleared in Hemo-De (Fisher Scientific,Pittsburgh, Pa.) and embedded in ParaplastPlus (Fisher Scientific).Tissue sections (10 μm thick) were mounted on SuperfrostPlus slides(Fisher Scientific). Section pretreatment and hybridization wereperformed according to (Lincoln et al., 1994, Plant Cell 6:1859-1876)except that proteinase K was used at 30 mg/ml and a two hourprehybridization step was included. Probe concentration of 50 ng/ml/kbwas used in the hybridization.

Slides were washed and the immunological detection was performedaccording to (Coen et al., 1990, Cell 63:1311-1322) with the followingmodifications. Slides were first washed 5 h in 5×SSC, 50% formamide.After RNase treatment slides were rinsed three times (20 min each) inthe buffer (0.5 M NaCl, 10 mM Tris-HCl pH 8.0, 5.0 mM EDTA). In theimmunological detection, antibody was diluted 1:1000, levamisole (240ng/ml) was included in the detection buffer, and after stopping thereaction in 10 mM Tris, 1 mM EDTA, sections were mounted directly toAqua-Poly/Mount (Polysciences, Warrington, Pa.).

6.2. Results

6.2.1 Characterization of the SCR Phenotype

The scarecrow mutant scr-1 was isolated in a screen of T-DNA transformedArabidopsis lines (Feldmann, K. A., 1991, Plant J. 1:71-82), as aseedling with greatly reduced root length compared to wild-type (Schereset al., 1995, Development 121:53-62). A second mutant scr-2 with asimilar phenotype was subsequently identified among T-DNA transformedlines. Analysis of co-segregation between the mutant phenotype andantibiotic resistance carried by the T-DNA indicated tight linkage forscr-1 and no linkage for scr-2 (see Experimental Procedures). Anantibiotic sensitive line of scr-2 was isolated and crossed with scr-1.The F2 progeny of this cross were all mutant and segregated 3:1 forantibiotic resistance confirming allelism (see Materials & Methods). Theprincipal phenotypic difference between the two alleles was that scr-1root growth was more retarded than that of scr-2, suggesting that it isthe stronger allele (FIG. 2A). For both alleles the aerial organsappeared similar to wild-type and the flowers were fertile (FIGS. 2A and2B). The progeny of backcrosses of scr-1 or scr-2 to wild-type plantssegregated 3:1 for the root phenotype for both alleles, indicating thateach mutation is monogenic and recessive.

Analysis of transverse sections through the primary root of seedlingsrevealed only a single cell layer between the epidermis and thepericycle (FIG. 2C) instead of the normal radial organization consistingof cortex and endodermis (FIG. 2D). This radial organization defect wasnot limited to the primary root, but was also present in secondary roots(FIG. 2E) and in roots regenerated from calli (FIG. 2F). Occasionallydefects were observed in the number of cells in the remaining cell layer(more than the invariant 8 found in wild-type). Abnormal placement ornumbers of epidermal cells were also observed (see FIG. 2E). Theseabnormalities were more frequently observed in scr-1 than in scr-2.Nevertheless, organization of the mutant root closely resembles that ofwild-type except for the consistent reduction in the number of celllayers. Because the endodermis and cortex are normally generated by anasymmetric division of the cortex/endodermal initial, this indicatesthat the primary defect in scr is disruption of this asymmetricdivision.

It has been shown that the radial organization defect in scr-1 firstappears in the developing embryo at the early torpedo stage andmanifests itself as a failure of the embryonic ground tissue to undergothe asymmetric division into cortex and endodermis (Scheres et al.,1995, Development 121:53-62). This defect extends the length of theembryonic axis which encompasses the embryonic root and hypocotyl. Otherembryonic tissues appear similar to wild-type (Scheres et al., 1995,Development 121:53-62). In seedling hypocotyls of the scarecrowphenotype, two cell layers instead of the normal three layers (twocortex and one endodermis) between epidermis and stele were found. Thiswould be the expected result of the lack of the division of theembryonic ground tissue. Similar results were obtained for scr-2. Hence,this mutant identifies a gene involved in the asymmetric division thatproduces cortex and endodermis from ground tissue in the embryonic rootand hypocotyl and from the cortex/endodermal initials in primary andsecondary roots.

6.2.2. Characterization of Cell Identity in SCR Roots

To understand the role of the Arabidopsis SCR gene in regulating thisasymmetric division, it was necessary to determine the identity of themutant cell layer. Tissue-specific markers were used to distinguishbetween several possibilities. The cell layer could have differentiatedattributes of either cortex or endodermis. Alternatively, it could havean undifferentiated, initial-cell identity or it could have a chimericidentity with differentiated attributes of both endodermis and cortex inthe same cell.

Transverse sections of scr-1 and scr-2 roots were assayed for thepresence of tissue-specific markers. The casparian strip, a depositionof suberin between radial cell walls, is specific to the endodermalcells and is believed to act as a barrier to the entry of solutes intothe vasculature (Esau, K. Anatomy of Seed Plants, New York: John Wiley &Sons, 1977, Ed. 2, pp. 1-550). Histochemical staining revealed thepresence of a casparian strip in the mutant cell layer (FIG. 3A, compareto wild-type, FIG. 3B). It is noted that in the vascular cylinder, thishistochemical stain also reveals the presence of lignin, indicating thepresence of differentiated xylem cells in mutant (FIG. 3A) and wild-type(FIG. 3B). Another marker of the differentiated endodermis is thearabinogalactan epitope recognized by the monoclonal antibody, JIM13(Knox et al., 1990, Planta 181:512-521). The mutant cell layer showedstaining with this antibody (FIG. 3C, compare with wild-type, FIG. 3B).As a positive control, the JIM7 antibody that recognizes pectin epitopesin all cell walls was used (FIGS. 3E and 3F). These results indicatethat the cell layer between the epidermis and the pericycle hasdifferentiated attributes of the endodermis.

As a marker for the cortex, the CCRC-M2 monoclonal antibody was used.This antibody recognizes a cell wall oligosaccharide epitope, found onlyon differentiated cortex and epidermis cells. In sections from thedifferentiation zone of scr-1 and scr-2, both cortex and epidermal cellsshowed staining (FIG. 4A and 4B) that was similar to that of wild-type(FIG. 4C). In scr-1, staining of both cell types was apparent, butstaining of cortex was somewhat weaker than wild-type. The positivecontrol used the CCRC-M1 monoclonal antibody which recognizes anoligosaccharide epitope found on all cells (FIGS. 4D-F).

With the CCRC-M2 antibody an interesting difference was observed betweenthe staining pattern of the mutants as compared to wild-type. Theappearance of this epitope correlates with differentiation in these twocell types. Normally, in sections close to the root tip there is nostaining. In sections higher up in the root, atrichoblasts (epidermalcells that do not make root hairs) stain. In sections from more matureroot tissue, all epidermal cells as well as cortex cells stain for thisepitope. In both scr-1 and scr-2, sections could be found in which allepidermal cells stained while there was little detectable staining ofcortex cells. Although not precisely identical to the wild-type stainingpattern, the fact that the mutant cell layer clearly stains for thiscortex marker indicates that there are cortex differentiated attributesexpressed in these cells.

Taken together, these results indicate that the mutant cell layer hasdifferentiated attributes of both the endodermis and cortex. Thepossibility that there has been a simple deletion of a cell type, orthat the resulting cell type remains in an undifferentiated initial-likestage can be ruled out. This result is consistent with a role for thescr gene in regulating this asymmetric division rather than a role indirecting cell specification. 6.2.3. Molecular Cloning of the SCR Gene

To further elucidate the function of the Arabidopsis SCR gene theinserted T-DNA sequences were used to clone the gene. Plant DNA flankingthe insertion site was obtained from scr-1 by plasmid rescue and used toisolate the corresponding wild-type genomic DNA. Several cDNA cloneswere isolated from a library made from silique tissue. Comparison of thesequence of the longest cDNA and the corresponding genomic regionrevealed an open reading frame (ORF) interrupted by a single smallintron. (FIG. 5A). A potential TATA box and polyadenylation signal thatmatched the consensus sequences for plant genes were also identified(Joshi, C. P., 1987, Nucl. Acids Res. 15:6643-6653); Heidecker &Messing, 1986, Ann. Rev. Plant Physiol. 37:439-466); Mogen et al., 1990,Plant Cell 2:1261-1272).

Comparison of the nucleotide sequence between the genomic clone and therescued plasmid placed the site of the T-DNA insertion in scr-1 at codon470 (FIGS. 5A and 5B). For scr-2, although no linkage was found betweenthe mutant phenotype and antibiotic resistance, DNA blot and PCRanalysis of antibiotic sensitive lines revealed the presence of T-DNAsequences that co-segregated with the mutant phenotype. The insertionposition in scr-2 was determined by cloning and sequencing the PCRproducts amplified from its genomic DNA using a combination of T-DNA andSCR specific primers at both sides of the insertion (FIG. 5B). In scr-2the T-DNA insertion point is at codon 605 (FIGS. 5A and 5B). To verifylinkage between the cloned gene and the mutant phenotype, we identifiedthe chromosomal location of both the scr locus and the SCR gene. To mapthe scr locus, molecular markers were used on F2 progeny of crossesbetween scr-2 (ecotype Wassilewskija, Ws) and Colombia (Col) WT. Theseplaced the scr locus at the bottom of chromosome III, approximately 0.5cM away from each of the two closest markers available, cdc2b and BGL1(Konieczny and Ausubel, 1993, Plant J. 4:403-410). To map the SCR gene,we identified a polymorphism between Col and Landsberg (Ler) ecotypesusing the SCR probe b (FIG. 5B). Southern analysis of 25 recombinantinbred lines (Jarvis et al., 1994, Plant Mol. Biol. 24:685-687) mappedthe cloned gene to the same location as the SCR locus on chromosome III.

The determination of the molecular defects in two independent allelesand the co-localization of the cloned gene and the mutant locus confirmsthat we have identified the SCR gene.

6.2.4. The SCR Gene has Motifs that Indicate it is a TranscriptionFactor

The Arabidopsis SCR gene product is a 653 amino acid polypeptide thatcontains several domains (FIG. 5B). The amino-terminus has homopolymericstretches of glutamine, serine, threonine, and proline residues, whichaccount for 44% of the first 267 residues. Domains rich in theseresidues have been shown to activate transcription and may serve such arole in SCR (Johnson et al., 1993, J. Nutr. Biochem 4:386-398). Acharged region between residues 265 and 283 has similarity to the basicdomain of the bZIP family of transcriptional regulatory proteins (FIG.5C) (Hurst, H. C., 1994, Protein Profile 1:123-168). The basic domainsfrom several bZIP proteins have been shown to act as nuclearlocalization signals (Varagona et al., 1992, Plant Cell 4:1213-1227),and this region in SCR may act similarly. This charged region isfollowed by a leucine heptad repeat (residues 291-322). A second leucineheptad repeat is found toward the carboxy-terminus (residues 436 to473). As leucine heptad repeats have been demonstrated to mediateprotein-protein interactions in other proteins (Hurst, H. C., 1994,Protein Profile 1:123-168), the existence of these motifs suggests thatSCR may function as a dimer or a multimer. The second leucine heptadrepeat is followed by a small region rich in acidic residues, alsopresent in a number of defined transcriptional activation domains(Johnson et al., 1993, J. Nutr Biochem 4:386-398). While each of thesedomains has been found within proteins that do not act astranscriptional regulators, the fact that all of them are found withinthe deduced SCR protein sequence indicates that SCR is a transcriptionalregulatory protein.

5 6.2.5. SCR is a Member of a Novel Protein Family

The Arabidopsis SCR protein sequence was compared with the sequences inthe available databases. Eleven expressed sequence tags (ESTs), ninefrom Arabidopsis, one from rice and one from maize, showed significantsimilarity to residues 394 to 435 of the SCR sequence, a regionimmediately amino-terminal to the second leucine heptad repeat (FIGS.15K-L). This region is designated the VHIID (amino acid residues 8-12 ofSEQ ID NO:12) domain. Subsequent analysis of these EST sequences hasrevealed that the sequence similarity extends beyond this region; infact, the similarity extends throughout the entire known gene products.The combination and order of the motifs found in these sequences do notshow significant similarity to the general structures of otherestablished regulatory protein families (i.e., bZIP, zinc finger,MADS-domain, and homeodomain), indicating that the SCR proteins comprisea novel family.

6.2.6. SCR is Expressed in the Cortex/Endodermal Initials and in theEndodermis

RNA blot analysis revealed expression of SCR in Arabidopsis siliques,leaves and roots of wild-type plants (FIG. 6A). No hybridization wasdetected to RNA from scr-1 plants (FIG. 6B, lane 2). This indicates thatscr-1 has a reduced level of RNA expression and may represent the nullphenotype. Hybridization to RNA species larger than the normal size weredetected in scr-2. This indicates that abnormal SCR transcripts are madein this allele, suggesting that functional but possibly altered proteinsmay be produced.

To determine if expression was localized to any particular cell type,RNA in situ was hybridization performed on sections of root tissue. Inmature roots, expression was localized primarily to the endodermis(FIGS. 7A and 7B). Expression appeared to start very close to or withinthe cortex/endodermal initials and continue up the endodermal cell fileas far as the section extended. Expression was also detected inlate-torpedo stage embryos in the endodermis throughout the embryonicaxis (FIG. 7C). Sense strand controls showed only backgroundhybridization (FIG. 7D).

To determine whether the localization of SCR RNA was regulated at thetranscriptional or post-transcriptional level, enhancer trap (ET) lineswere prepared and examined in which the β-glucuronidase (uid-A or GUS)coding sequence with a minimal promoter was expressed in the rootendodermis. (See Section 7, infra). Restriction fragment lengthpolymorphisms were observed when DNA from one of these lines, ET199 andwild-type were probed with SCR. PCR and sequence analysis confirmed thatthe enhancer-trap construct had inserted approximately 1 kb upstream ofthe SCR start site and in the same orientation as that of SCRtranscription.

In mature roots, expression in ET199 whole mounts showed a similarpattern to that of the in situ hybridizations, with the strongeststaining present in endodermal cells (FIG. 7E). Transverse sectionsindicated that expression was primarily in endodermal cells in theelongation zone (FIG. 7F). Longitudinal sections through themeristematic zone revealed that expression could be detected in thecortex/endodermal initial (FIG. 7G). Of particular interest was therestriction of expression to the endodermal daughter cell after thepericlinal division (FIG. 7G). This indicated that the expressionpattern observed in the in situ analysis was not due topost-transcriptional partitioning of SCR RNA. Rather, it suggests thatafter the periclinal division of the cortex/endodermis initial only oneof the two cells is able to transcribe SCR RNA.

6.3. Discussion

6.3.1. The SCR Gene Regulates an Asymmetric Division Required for RootRadial Organization

The formation of the cortex and endodermal layers in the Arabidopsisroot requires two asymmetric divisions. In the first, an anticlinaldivision of the cortex/endodermal initial generates two cells withdifferent developmental potentials. One will continue to function as aninitial, while the other undergoes a periclinal division to generate thefirst cells in the endodermal and cortex cell files. This secondasymmetric division is eliminated in the scarecrow mutant, resulting ina single cell layer instead of two. The scr mutation appears to havelittle effect on any other cell divisions in the root indicating that itis involved in regulating a single asymmetric division in this organ.Several other mutations have been characterized that appear to affectspecific cell division pathways in Arabidopsis. These include knolle(kn) in which formation of the epidermis is impaired (Lukowitz et al.,1996, Cell 84:61-71), wooden leg (wol) in which vascular cell divisionis defective (Scheres et al., 1995, Development 121:53-62) and fass (fs)in which there are supernumerary cortex and vascular cells (Scheres etal., 1995, Development 121:53-62); Torres Ruiz & Jurgens, 1994,Development 120:2967-2978). Only in the case of scr and short-root (shr)mutants has it been shown that the defect is in a specific asymmetricdivision.

Mutational analyses in several organisms have revealed that the genesthat regulate asymmetric divisions can be specific to a single type ofdivision or can affect divisions that are not clonally related (Horvitz& Herskowitz, 1992, Cell 68:237-255). In most cases, these mutationsresult in the formation of two identical daughter cells with similardevelopmental potentials (Horvitz & Herskowitz, 1992, Cell 68:237-255).Both resulting cells have the identity of one or the other of the normaldaughter cells, an example of which is the swi⁻mutation in S. cerevisiae(Nasmyth et al., 1987, Cell 48:579-587). However, there are alsoexamples of mutations that result in the formation of chimeric celltypes such as the ham-1 mutation in C. elegans (Desai et al., 1988,Nature 336:638-646).

6.3.2. SCR Involvement in Cell Specification or Cell Division

Genes that regulate asymmetric cell divisions can be divided into thosethat specify the differentiated fates of the daughter cells and thosethat function to effect the division of the mother cell (Horvitz &Herskowitz, 1992, Cell, 68:237-255). The aberrant cell layer formed inthe scr mutant has differentiated features of both endodermal and cortexcells. Thus, scr is in the rare class of asymmetric division mutants inwhich a chimeric cell type is created. The ability to expressdifferentiated characteristics of cortex and endodermal cells impliesthat the differentiation pathways for both these cell types are intactand do not require the functional SCR gene. This indicates that SCR isinvolved primarily in regulating a specific cell division, and that thecorrect occurrence of this division can be unlinked from cellspecification. This is in contrast to the shr mutant, in which thepericlinal division of the cortex/endodermal initial also fails to occurand the resulting cell lacks endodermal markers (Benfey et al., 1993,Development 119:57-70) and has cortex attributes. A genetic analysis wasused to address the function of SHR and SCR in the asymmetric divisionof the cortex/endodermal initial. Placing mutants of each of these genesin a fs mutant background asked whether the supernumerary cell divisionscharacteristic of fs were sufficient to restore normal cell identities(Scheres et al., 1995, Development 121:53-62). In the shr,fs doublemutant there were additional cell layers but no endodermal, indicatingthat the SHR gene has a role in specifying cell identity. In the scr,fsdouble mutant no alteration in cell identity was observed as compared tofs (Scheres et al., 1995, Development 121:53-62). Taken together withthe cell marker analysis presented herein, these results are consistentwith a role for SCR in generating the division of the mother cell whilethe SHR gene may be involved in specifying the fate of the endodermaldaughter.

6.3.3. A Role for SCR in Embryonic Development

At least one additional cell division appears to be affected in the scrmutant. During embryonic development, the ground tissue does not divideto form the endodermal and cortex layers of the embryonic root andhypocotyl. As shown herein, expression of SCR was detected in theendodermal tissue throughout the embryonic axis shortly after thisdivision occurs. Thus, SCR may play a direct role in regulating boththis division and the division of the cortex/endodermal initial in theroot apical meristem. Alternatively, the radial organization establishedin the embryo may somehow act as a template that directs the division ofthe cortex/endodermal initial, thus perpetuating the pattern. This isconsistent with the finding in the scr mutant that the aberrant patternestablished in the embryo is perpetuated in the primary root. It is alsoconsistent with a recent study in which the daughter cells of thecortex/endodermal initial were laser ablated (van den Berg et al., 1995,Nature 378:62-65). When a single daughter cell was ablated, it wasreplaced by a cell that followed the normal asymmetric division pattern.When three adjacent daughter cells were ablated, the central initialdivided anticlinally but failed to perform the periclinal division (vanden Berg et al., 1995, Nature 378:62-65). This provided evidence thatinformation from mature cells is required for the correct divisionpattern of cortex/endodermal initials suggesting a “top down” transferof information. However, the absence of a cell layer in lateral rootsand callus-derived roots of the scr mutant suggests that embryo eventsare not unique in their ability to establish radial organization.Rather, these observations implicate SCR in regulating both embryonicand post-embryonic root radial organization.

5 6.3.4. Tissue-Specific Expression of SCR is Regulated at theTranscriptional Level

Although not intending to be limited to any theory or explanationregarding the mechanism of SCR action, the cloning of the gene and theexpression pattern provide some clues as to the role of SCR in theregulation of a specific asymmetric division. The SCR gene is expressedin the cortex/endodermal initial, but immediately after division isrestricted to the endodermal lineage. A similar pattern is seen in theET199 enhancer trap line in which SCR regulatory elements are inproximity to a GUS gene, indicating that SCR restriction to theendodermal cell file is due to differential regulation of expression ofthe SCR gene in this cell and the first cell in the cortex file. Anothermarker line in which expression of GUS is detected only in the cortexdaughter cell provides a control for differential degradation of GUS RNAor protein. Thus, partitioning of SCR RNA as a means of achieving thissegregation of expression can be ruled out. What remains to bedetermined is whether this difference in transcriptional activity of thetwo daughter cells is due to internal polarity of the mother cell priorto division such that cytoplasmic determinants are unequallydistributed, or to external polarity that influences cell fate afterdivision. Since SCR is expressed prior to cell division, an attractivehypothesis is that it is involved in establishing polarity in thecortex/endodermal initial. The sequence of the SCR protein stronglysuggests that it acts as a transcription factor. Hence, it may act toregulate the expression of other genes essential for the establishmentof unequal division. Alternatively, it is conceivable that it could playa role in creating an external polarity that provides a signal to divideasymmetrically. Its expression in more mature endodermal cells isconsistent with a role in “top-down” signaling.

6.3.5. A New Family of Transcriptional Regulators

Analysis of eighteen EST clones found in the GenBank database revealsthat the proteins they encode share a high degree of homology withArabidopsis SCR protein. See Table 1 and FIGS. 15A-S. Further sequenceanalysis of the encoded proteins indicate that a high degree of sequencesimilarity extends from at least the highly conserved VHIID (amino acidresidues 8-12 of SEQ ID NO:12) domain to the carboxy-terminus of thegene products. Comparison of the amino termini of these proteins isprecluded by the fact that the ESTs are incomplete. The high degree ofsimilarity among these proteins, in combination with the motifs observedin the SCR protein (homopolymeric motifs, two leucine heptad repeats anda bZIP-like basic domain that may also function as a nuclearlocalization sequence) indicates that these proteins form a novel classof regulatory proteins.

The insertion sites of the T-DNA in the two scr mutant alleles raisedthe possibility that the mutant phenotype was due to the production oftruncated proteins. Northern blot analysis indicated SCR RNA isundetectable in scr-1. This suggests that the phenotype is either thenull, or due to highly reduced RNA expression. In scr-2, an alterationin RNA size was detected which would be consistent with the presence ofa functional and possibly truncated protein. This could provide anexplanation for the observation that scr-2 appears to be the weakerallele.

7. EXAMPLE 2 Enhancer Trap Analysis of Root Development

An enhancer trap system was used in order to provide a more detailedmolecular analysis of gene expression in lateral root patterning anddevelopment in Arabidopsis thaliana. A new collection of marker linesthat express β-glucuronidase (GUS) activity in a cell-type specificmanner in each of the cells of the root was generated. These lines allowdifferentiation of cells to be monitored based on molecularcharacteristics. One of these marker lines, ET199, resulted from theintegration of the GUS cassette in proximity to an SCR enhancer. Theresults described below demonstrate that transcriptional activation ofthe SCR gene plays an important role in root development in Arabidopsis,and that SCR gene transcriptional regulatory elements can express atransgene in a developmentally and tissue specific manner.

7.1. Materials and Methods

7.1.1. Plant Growth Conditions

Arabidopsis seeds from NO-O and Columbia ecotypes were sterilized andsown on MS plates containing 4.5% sucrose. Plates were orientedvertically and maintained under 18 hours light, 6 hours dark cycle.

7.1.2. Histology and Gus Staining

For observation of lateral roots, roots were removed from plates andinfiltrated in 25% glycerol for several hours to overnight. Roots werethen mounted in 50% glycerol. Whole seedlings were stained for GUSactivity for up to three days in the following solution: 1× GUS buffer,20% methanol, 0.5 mg/ml X-Glu. Addition of methanol greatly improves thespecificity and reproducibility of staining. Staining solution was madefresh from a 10× buffer (1 M Tris pH7.5, 290 mg NaCl, 66 mg K₃Fe(CN)₆)that was stored for no more than one week. Stained roots were cleared inglycerol and mounted as above. All samples were observed using Nomarskioptics on a Leitz Laborlux S microscope. Photographs were taken using aLeitz MPS52 camera, and images were scanned into Adobe Photoshop tocreate figures. In some cases the intensity of the blue color wasincreased.

7.1.3. Construction of Enhancer Trap Lines

Plant Cloning Vector (PCV) (Koncz et al., 1994, Specialized vectors forgene tagging and expression studies, in Plant Molecular Biology Manual,Gelvin & Schilperoort, eds., Vol. B2, pp. 1-2, Kluover Academic Press,Dordrecht, The Netherlands) contains a Bam HI site immediately adjacentto the T-DNA right border sequence. The β-glucuronidase gene fused tothe TATA region (−46 to 78) of the CaMV 35S promoter was introduced intothis site (Benfey et al., 1990, EMBO J. 9:1677-1684). 350 transgeniclines were generated by Agrobacterium mediated root transformation(Marton & Browse, 1991, Plant Cell Reports 10:235-239), and 4independent lines from each transformant were screened for GUS activityin the root.

7.2. Results

7.2.1. Differentiation in the LRP

The marker lines described above reflect patterns of gene expressionthat are specific to individual root cell types. There are no readilyapparent mutant phenotypes in any of these lines. Therefore, they can beused to analyze the differentiation state of the cells during normaldevelopment of the lateral root primordial (LRP). If there are stages atwhich the pericycle cells proliferate in the absence of patterning, itcan be expected that all cells would be identical with none expressingdifferentiated characteristics. In contrast, organization of the LRPwould be reflected in differential patterns of GUS gene expression, withcertain cells beginning to turn on transcription from differentiatedcell-type specific promoters (i.e., those that drive GUS expression inthe enhancer trap lines).

The process of lateral root formation is divided into the followingseven stages:

Stage I: The LRP is first visible as a set of pericycle cells that areclearly shorter in length than their neighbors, having undergone aseries of anticlinal divisions. Laskowski et al., 1995, Dev.121:3303-3310 predict that there are approximately 4 founder pericyclecells involved. In the longitudinal plane, these divisions result in theformation of 8-10 small cells, which enlarge in a radial direction.

Stage II: A periclinal division occurs that divides the LRP into twolayers (Upper Layer (UL) and Lower Layer (LL)). Not all the smallpericycle-derived cells appear to participate in this division—typicallythe most peripheral cells do not divide. Hence, as the UL and LL cellsexpand radially the domed shape of the LRP begins to appear.

Stage III: The UL divides periclinally, generating a three layerprimordium comprised of UL1, UL2 and LL. Again, some peripheral cells donot divide, creating peripheral regions that are one and two cell layersthick. This further emphasizes the domed shape of the LRP.

Stage IV: The LL divides periclinally, creating a total of four celllayers (UL1, UL2, LL1, LL2). At this stage the LRP has penetrated theparent endodermal layer.

Stage V: The central cells in LL2 undergo a number of divisions thatpush the overlying layers up and distort the cells in LL1. Thesedivisions are difficult to visualize at this stage, but clearly form aknot of mitotic activity. The LRP at this stage is midway through theparent cortex. The outer layer contains 10-12 cells.

Stage VI: This stage is characterized by several events. The fourcentral cells of UL1 divide periclinally. This division is particularlyuseful in identifying the median longitudinal plane in the enlargingLRP. At this point there are a total of twelve cells in UL1, four in themiddle that have undergone the periclinal division and four on eitherside. In addition, all but the most central cells of UL2 undergo apericlinal division. At this point the LRP has passed through the parentcortex layer and has penetrated the epidermis. The central cellsapparently derived from LL2 have a distinct elongated shapecharacteristic of vascular elements.

Stage VII: As the primordium enlarges it becomes difficult tocharacterize the divisions in the internal layers. However, the cells inthe outermost layer can still be seen very clearly. All of these cellsundergo a anticlinal division, resulting in 16 central cells (8 cells ineach of two layers) flanked by 8-10 cells on each side. We refer to thisas the 8-8-8 cell pattern. The LRP appears to be just about to emergefrom the parent root.

7.2.2. Marker Lines

An enhancer trapping cassette was generated by fusing the GUS codingsequence to the minimal promoter of the 35S promoter from CaMV. Thisminimal promoter does not produce a detectable level of GUS expression.However, its presence allows other upstream elements to direct GUSexpression in a developmental and/or cell-specific manner (Benfey etal., 1990, EMBO J. 9:1677-1684). The use of a minimal promoter insteadof a promoterless construct allows GUS expression to occur even if theenhancer trap cassette inserts at a distance from the coding region.Since the insert does not have to be within the structural gene, thereare often no mutations generated in the enhancer trap lines. The minimalpromoter:GUS construct was cloned immediately adjacent to the T-DNAright border sequence of PCV (Koncz et al., supra) and introduced intoArabidopsis. 350 independent lines were generated and analyzed for GUSactivity in the root. The following lines most clearly define each celltype. All of the lines were generated through enhancer trapping, asdescribed herein, below, except for CorAX92 (Dietrich et al., 1992,Plant Cell 4:1371-1382) and EpiGL2:GUS (Masucci et al., Dev.122:1253-1260) which are transgenic plants that contain cell-typespecific promoters fused to the GUS gene.

Ste05—expresses GUS in the stele including the pericycle layerthroughout primary and lateral roots. At the root tip, staining becomesweaker in the elongation zone; therefore, it is likely that onlydifferentiated stele cells express GUS activity. Stelar GUS expressionis also seen in aerial parts of the plant.

End195—expresses GUS in the endodermis of primary and lateral roots.Staining can be seen most clearly in the cells in the meristematicregion of the root, although overstaining shows that more mature cellsalso express some GUS activity. It appears that there is no staining inthe cortex/endodermal initial, but staining is evident in the firstdaughter cell of this initial. GUS expression is also seen at the baseof young leaves and in the stipules.

ET199—expresses GUS in the endodermis of primary and lateral roots,again most clearly in cells in the meristematic region. Unlike End195,staining in ET199 appears to continue down to the cortex/endodermalinitial and, in younger roots, even into the cells of the quiescentcenter. Expression in the aerial parts of the plant is detectable in theyoung leaf primordia.

CorAX92—This line was generated by fusing the 5′ and 3′ sequences from acortex specific gene isolated from oilseed rape to the GUS reporter gene(Dietrich et al., Plant Cell 4:1371-1382). Expression is limited to thecortex layer, extending to but not including the cortex/endodermalinitial. Staining is also apparent in the petioles and leaf blades ofexpanded leaves.

EpiGL2:GUS—This line was generated by fusing the GL2 promoter to the GUSgene (Masucci et al., Dev. 122:1253-1260). Expression is seen in thenon-hair forming epidermal cells (atrichoblasts). Staining is seen nearthe root tip, but it is difficult to determine if it includes theepidermal initial. Staining is also seen in the trichomes, leafprimordia, and the epidermis of the hypocotyl and leaf petioles.

CRC219—This line shows staining in the columella root cap only.

LRC244—This line shows staining in the lateral root cap only.

RC162—This line shows staining in both the lateral and columella rootcaps.

Two marker lines show differential staining at very early stages of LRPdevelopment. One of these, ET199, presents a complex and dynamic patternof expression. Staining is first apparent at stage II in only the fourcentral cells of the UL. At stage III staining is strongest in thecentral cells of UL2. As the LRP reaches stage V the staining remainsstrongest in the central 2-4 cells of UL2. By stage VI staining alsobegins to extend into the newly formed endodermal layer, and staining inboth the central cells and endodermis persists beyond emergence of thelateral root.

Another line, LRB10 (lateral root base), does not express GUS in theprimary root tip. Staining in the LRP is seen at stage I, and at stageII all the cells of the UL and LL are stained. However, by stage IV andV only the cells at the periphery of the LRP are still expressing GUS.As the LRP develops, these cells continue to stain, although lessintensely, resulting in a ring of GUS expressing cells at the base ofthe LR.

LRB10 and ET199 clearly demonstrate non-identity between the cells atvery early stages, stage IV in the case of LRB10 and within the UL atstage II in ET199. In addition, although it is difficult to identify thenature of the cells that correspond to the observed staining pattern inLRB10 and the early staining cells of ET199, post-emergent lateral rootsshow analogous staining in these lines, suggesting that the stainedcells are already expressing markers that reflect their differentiatedcell fates. Hence, these observations suggest a very early onset ofdifferentiation in the cells of the LRP.

7.2.3. ET199 Provides Evidence for the Role of SCR in Plant Development

Fortuitously, it was discovered that the GUS cassette in ET199 describedSection 7.2.2, above, is situated approximately 1 kb upstream from theSCR gene. The SCR cDNA was labelled and used to probe genomic DNA fromWT and ET199 plants. The band pattern seen in the Southern wascompletely consistent with a T-DNA inserted 1 kb upstream of theputative SCARECROW start site. Subsequently, a DNA fragment was PCRamplified using a primer within the T-DNA and a primer within SCARECROW.The size of this fragment was also consistent with the predictedinsertion site. Partial sequencing of the PCR fragment confirmed thepresence of SCARECROW sequence. Mutants in the SCR gene are completelylacking one of the radial layers between the epidermis and pericycle inboth primary and lateral roots, due to the absence of specific celldivision during embryogenesis and of the cortex/endodermal initialduring post-embryonic growth. The expression pattern (described inSection 7.2.2., above) that was observed in the central cells of thedeveloping LRP of ET199 provide strong evidence that the cells in thisregion are involved in the establishment of the meristematic initials.More importantly, these results demonstrate that transcriptionalactivation of the SCR gene plays a major role in the development of theArabidopsis LRP. Furthermore, these results demonstrate that a transgenecan be expressed under the control of SCR gene transcriptionalregulatory elements in a developmental and tissue-specific manner.

8. EXAMPLE 3 Activity of Arabidopsis SCR Promotor in Transgenic Roots

The expression pattern of Arabidopsis SCR has been determined byanalysis of an enhancer trap line, ET199, in which a GUS coding regionwith a minimal promoter was fortuitously inserted 1 kb upstream of theSCR coding region (see supra). In ET199 plants, GUS expression isdetected in the endodermis, endodermal initials and sometimes in thequiescent center (QC) of the root. See supra and Malamy and Benfey,1997, Dev. 124:33-44. This expression pattern of SCR in the primary roothas been confirmed by in situ analysis (See supra and Di Laurenzio etal., 1996, Cell 86:423-433).

The following experiments demonstrate that 2.5 kb of 5′ sequenceupstream of the Arabidopsis SCR coding region is sufficient to conferSCR expression pattern to a heterologous gene. The 5′ sequence used inthese studies starts from the Hind III site approximately 2.5 kbupstream of the ATG initiation site and extends 3′ downstream to thebase pair immediately upstream of the ATG initiation site (see FIG. 14).This 5′ sequence was fused to a GUS coding sequence. The resulting SCRpromoter::GUS construct was incorporate into an Agrobacterium vector,which was used to transform and generate transgenic roots using standardprocedures.

A large number of roots were regenerated. They show GUS staining patternthat is similar to the SCR expression pattern in ET199 plants (FIG. 19,Panel f). Since organs regenerated from callus often have an abnormalmorphology, transgenic roots were transferred to liquid culture. Rootsgrown in liquid culture appeared morphologically normal and showed GUSexpression in the endodermis, endodermal initial and QC (FIG. 19, Panelg), similar to the expression pattern of SCR seen in the enhancer trapline ET199. These results indicate that the 2.5 kb region upstream ofthe SCR start site is sufficient to confer the SCR expression pattern inthe root.

The expression of the SCR promoter::GUS construct was also examined inscr mutant background. The scr mutant has an altered root organization(see, supra). Whereas the wild-type root of Arabidopsis has fourdistinct cell layers surrounding the vascular tissue, the roots of scrmutant have only three.

Transgenic roots of the scr mutant were generated that contained a SCRpromoter::GUS construct. As in the wild-type, a large number oftransgenic roots were formed that had detectable GUS expression (FIG.20, Panel a). These roots were shorter than wild-type regenerated roots,consistent with the shorter root phenotype of the scr mutant.

Additional transgenic root experiments demonstrated that the SCR geneunder control of its own promoter can rescue the scr mutant phenotype.Transgenic scr roots were generated that contained the full length SCRgene under the control of its own promoter. The length of transgenicroots containing the construct were longer than those of the scr mutant,indicating that the introduced SCR gene partially rescued the mutant.Whereas scr regenerated roots that carried the SCR promoter::GUSconstruct were very short (FIG. 21, Panel a; and FIG. 20, Panel a),roots transformed with the SCR promoter and coding region werenoticeably longer (FIG. 21, Panel b). The difference was even moreobvious in liquid culture, in which scr mutant roots remained short(FIG. 21, Panel c), while SCR gene complemented scr mutant roots werelong and resembled wild-type roots (FIG. 21, Panel d).

Anatomical studies of the regenerated roots confirmed the ability of theSCR promoter::SCR gene construct to rescue the scr mutant phenotype.Whereas regenerated roots of scr mutant were missing an internal layer(FIG. 21, Panel e), the scr mutant roots that were transformed with theSCR promoter::SCR gene construct had a radial organization thatresembled wild-type root (FIG. 21, Panel f).

9. EXAMPLE 4 Isolation SCR Sequences Using PCR-cloning Strategy

Based on the comparison of the sequences of SCR paralogs in Arabidopsis,degenerate primers SCR3AII, SCR5AII and SCR5B were designed and used inPCR amplification of SCR sequences from genomic DNA of various plantspecies. The amplification was performed according to conditiondescribed in Section 5.1.1., supra, using DNA isolated from maize plantsgrown from a commercial seed mixture. Amplification products (104 bpfragment for the SCR5B+SCR3AII primer combination; 146 bp fragment forthe SCR5AII+SCR3AII primer combination) were obtained, and each clonedinto a T/A vector (Invitrogen, San Diego, Calif.) and sequenced. Two ofthe three different types of clones obtained had deduced amino acidsequences that were very similar to a part of the Arabidopsis SCRprotein (i.e., approximately 90% identity), suggesting that theyrepresent parts from two different alleles of the maize SCR gene (i.e.,ZCR gene). The two clones each had only two conservative changes intheir nucleotide sequence.

The 146 bp amplification product, ZmScl1, was subsequently used as aprobe for screening of a genomic library generated in lambda BlueSTARvector (NOVAGEN) from maize (HiII line) genomic DNA. The screening wasperformed according to the standard procedures described in Genius™System User's Guide For Membrane Hybridization (Boehringer-Mannheim):The probe was a single-strand DNA molecule corresponding to the ZmScl1fragment produced by PCR (Genius, Boehringer-Mannheim). Hybridizationwas performed according to recommendations of the manufacturer's manual(Boehringer-Mannheim). Prehybridization was for 2 hr in 50% formamidehybridization solution at 42° C. Hybridization was overnight at 42° C.with 200 ng/ml probe concentration. Filters were washed twice at roomtemperature in 2×SSC, 0.1% SDS for 5 min, and for stringent washing at65° C. in 0.5×SSC, 0.1% SDS twice for 15 min.

A positive clone was identified. The clone contained a 13 kb insert,which was subcloned into a plasmid vector. The resulting plasmid wasdesignated pZCR. A 5 kb Eco RI fragment containing the maize SCR (ZCR)sequence was subcloned and sequenced. The nucleotide sequence of theregion containing a partial ZCR coding sequence is shown in FIG. 17A andthe corresponding deduced amino acid sequence is shown in FIG. 17B. TheZCR protein contain a segment that is highly homologous to acorresponding segment in the Arabidopsis SCR protein (FIG. 17B). Thissegment is flanked by segments of low homology. Thus, it is possiblethat the genomic clone of ZCR is a composite clone, containing sequencesthat are not ZCR sequences.

The deduced ZCR protein sequence was aligned with that of ArabidopsisSCR protein. The comparison revealed new conserved sites in the SCRcoding sequence which were used to design new, more specific PCR primers(i.e., 1F, 1R, and 4R) for use in amplification of SCR sequences fromyet other plant species.

Using combinations of primers 1F+1R and 1F+4R, PCR amplification wereperformed as described in section 5.1.1. Two DNA of expected size wereobtain from soybean: a 247 bp DNA from the 1F+1R primer combination anda 379 bp DNA from the 1F+4R primer combination. A DNA of expected size(247 kb) was obtained from carrot and spruce when their genomic DNA wasamplified using 1F+4R primer combination. The nucleotide sequences ofthe 379 kb soybean DNA (SRPg1), the 247 kb DNA from carrot (SRPd1) andspruce (SRPp1) are shown in FIGS. 16K-M. The corresponding deduced aminoacid sequences of these amplified sequences are shown in FIG. 18.Comparison of these partial SCR coding sequences indicate this approachisolated DNA sequences that encode SCR proteins with amino acidsequences that are very similar but not identical to a segment ofArabidopsis SCR protein (see FIG. 18).

10. EXAMPLE 5 Expression Pattern of Maize ZCR Gene in Root Tissue

These experiments examined the expression pattern of ZCR in the primaryroot and quiescent centers of maize root. The expression pattern wasdetermined by in situ hybridization using a ZCR RNA probe, correspondingto an amino acid segment region that is highly homologous to acorresponding segment of the Arabidopsis SCR protein. The experiment wascarried out as follows. Restriction fragments containing the maize ZCRsequence were isolated from pZCR and subcloned into a pbluescript vectorfor in vitro transcription. The probe was synthesized using conditionsdescribed in the Genius Dig RNA labeling kit. The pBluescript plasmidwas linearized, and 1 μg was used as a template to synthesizedigoxigenin-labeled RNA using the T7 polymerase. The RNA probe wassubjected to mild alkali hydrolysis by heated at 60° C. for 1 hr in 100mM carbonate buffer (pH 10.2) to yield a probe size of approximately0.15 kb. Probe concentration for hybridization was optimized at 1μg/ml/kb. In situ hybridization of root tips from 48 to 72 hr-old maizeseedlings or excised quiescent centers (QCs) of roots were carried outfollowing procedures described in Section 6.1.6., supra.

The results show that ZCR expression in maize primary roots is localizedto a file of cells that is identified as the endodermal layer. Theexpression pattern continues in a single uninterrupted file through theQC which consists of approximately 1000-1500 cells (FIG. 22).

In two-week old regenerating QCs, ZCR expression is found in a file ofcells extending through the newly formed apex. Thus, the regeneratedroots exhibits a ZCR expression pattern that is similar to that seen inthe primary root, even though the root apex does not contain the normalarrangement of cell files at this stage.

ZCR expression during regeneration of the root apex was also examined.In the initial stages of regeneration, cell proliferation occurs to fillin the removed tissue and begins to regenerate the basic shape of theroot tip. All cells on the blunt edge of the root appears to contributeto the new population of cells. The ZCR expression pattern indicatesthat molecular signals are differentially present in these cells at anearly stage in regeneration. The gene appears to be diagnostic of cellsthat are preparing to undergo asymmetrical division in order tore-establish the normal organization of the root apex from the largeundifferentiated cells. The results indicate that ZCR expression isrequired for pattern formation since it is expressed prior to thegeneration of any specific anatomical pattern in the newly formed nottissue.

11. EXAMPLE 6 Expression Pattern of ZCR Gene in Soybean Roots and RootNodules

SCR expression in soybean roots and nodules was examined using in situhybridization with a SCR probe. The procedure used were as described inSections 6.1.6. and 11.

In primary roots, SCR is expressed in the endodermis. Expression wasalso found in cells at the root tip that are located at the distal endof the endodermal cell files. In soybean nodules, expression of SCR wasdetected in the peripheral tissue at the site of developing vascularstrands. At later stages of vascular development within the nodule, SCRexpression was found flanking the vascular tissue. These resultsindicate that SCR is involved in regulating vascularization in thenodule by contributing to the radial organization that is required togenerate endodermis. These findings indicate that SCR promoter may beused to express proteins in a highly tissue-specific manner in soybeannodules. One application is to use SCR promoter to engineer nodulesthrough production of components in a tissue-specific manner. Anotherapplication is that modification of the expression of SCR could enhancenodule activity by improving vascularization and/or the number ofendodermal layers.

12. EXAMPLE 7 SCR Expression Affects Gravitropism of Aerial Structures

In addition to being defective in specific embryonic and postembryonicmeristematic divisions, both the scr and the shr mutants have shootsthat exhibit severely defective gravitropism. Complementation analysisshowed that scr is allelic to a sgr (shoot gravitropism) mutant, sgr1.Four mutant alleles of SCR (i.e., scr1, scr2, sgr1-1 and sgr1-2) havebeen identified. All four of these mutants have normal root gravitropismand defective shoot gravitropism.

Etiolated hypocotyls of scr mutants placed on their sides do not respondto gravity even after 3 hr. Similar behaviors were observed with theinflorescence stems of sgr1-1 mutant, which do not curve upwards evenafter two days on their sides. In contrast, the roots of these plantsrespond rapidly to the change in orientation with the same kinetics asthe wild type. Thus, mutations in the SCR gene lead to a radial patterndeficiency in the root but have no effect on root gravitropism.

Comparable results were also obtained for shr roots and for hypocotylsand inflorescence stems, i.e., data indicate that shr shows normal rootgravitropism but almost no stem gravitropism.

13. DEPOSIT OF MICROORGANISMS

The following microorganisms have been deposited in accordance with theterms of the Budapest Treaty with the American Type Culture Collection;12301 Parklawn Drive, Rockville, Md. 20852, U.S.A., on the datesindicated:

Accession Microorganism Clone No. Date DH5α pGEX-2 TK⁺ 98031 April 26,1996 (pLIG 1-3/ Sac + MOB 1 Sac) DH5α pNYH1 (Zm-sc11b) 98032 April 26,1996 DH5α pNYH2 (Zm-sc11) 98033 April 26, 1996 DH5α pNYH3 (Zm-sc12)98034 April 26, 1996 DH5α pZCR April 18, 1997

Although the invention is described in detail with reference to specificembodiments thereof, it will be understood that variations which arefunctionally equivalent are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings such modificationsare intended to fall within the scope of the appended claims.

Various publications are cited herein, each of the disclosures of whichis incorporated by reference in its entirety.

79 1 2163 DNA Plant 1 ccttatttat aaccatgcaa tctcacgacc aacaacccttcaatctccat ggcggaatcc 60 ggcgatttca acggtggtca acctcctcct catagtcctctgagaacaac ttcttccggt 120 agtagcagca gcaacaaccg tggtcctcct cctcctcctcctcctccttt agtgatggtg 180 agaaaaagat tagcttccga gatgtcttct aaccctgactacaacaactc ctctcgtcct 240 cctcgccgtg tctctcacct tcttgactcc aactacaatactgtcacacc acaacaacca 300 ccgtctctta cggcggcggc tactgtatct tctcaaccaaacccaccact ctctgtttgt 360 ggcttctctg gtcttcccgt ttttccttca gaccgtggtggtcggaatgt tatgatgtcc 420 gtacaaccaa tggatcaaga ctcttcatct tcttctgcttcacctactgt atgggttgac 480 gccattatca gagaccttat ccattcctca acttcagtctctattcctca acttatccaa 540 aacgttagag acattatctt cccttgtaac ccaaatctcggtgctcttct tgaatacagg 600 ctccgatctc tcatgctcct tgatccttcc tcttcctctgacccttctcc tcaaactttc 660 gaacctctct atcagatctc caacaatcct tctcctccacaacagcaaca gcagcaccaa 720 caacaacaac aacagcataa gcctcctcct cctccgattcagcagcaaga aagagaaaat 780 tcttctaccg atgcaccacc gcaaccagag acagtgacggccactgttcc cgccgtccaa 840 acaaatacgg cggaggcttt aagagagagg aaggaagagattaagaggca gaagcaagac 900 gaagaaggat tacaccttct cacattgctg ctacagtgtgctgaagctgt ctctgctgat 960 aatctcgaag aagcaaacaa gcttcttctt gagatctctcagttatcaac tccttacggg 1020 acctcagcgc agagagtagc tgcttacttc tcggaagctatgtcagcgag attactcaac 1080 tcgtgtctcg gaatttacgc ggctttgcct tcacggtggatgcctcaaac gcatagcttg 1140 aaaatggtct ctgcgtttca ggtctttaat gggataagccctttagtgaa attctcacac 1200 tttacagcga atcaggcgat tcaagaagca tttgagaaagaagacagtgt acacatcatt 1260 gacttggaca tcatgcaggg acttcaatgg cctggtttattccacattct tgcttctaga 1320 cctggaggac ctccacacgt gcgactcacg ggacttggtacttccatgga agctcttcag 1380 gctacaggga aacgtctttc ggatttcaca gataagcttggcctgccttt tgagttctgc 1440 cctttagctg agaaagttgg aaacttggac actgagagactcaatgtgag gaaaagggaa 1500 gctgtggctg ttcactggct tcaacattct ctttatgatgtcactggctc tgatgcacac 1560 actctctggt tactccaaag gtaaaataaa cattaccttttaatcactct ttatctataa 1620 attattttaa gattatatag gaaagatatg ttctaaaaagctggcttttt tggttaatga 1680 ttggggaatg aacagattag ctcctaaagt tgtgacagtagtggagcaag atttgagcca 1740 cgctggttct ttcttaggaa gatttgtaga ggcaatacattactactctg cactctttga 1800 ctcactggga gcaagctacg gcgaagagag tgaagagagacatgtcgtgg aacagcagct 1860 attatcgaaa gagatacgga atgtattagc ggttggaggaccatcgagaa gcggtgaagt 1920 gaagtttgag agctggaggg agaaaatgca acaatgtgggtttaaaggta tatctttagc 1980 tggaaatgca gctacacaag cgactctact gttgggaatgtttccttcgg atggttacac 2040 tttggttgat gataatggta cacttaagct tggatggaaagatctttcgt tactcactgc 2100 ttcagcttgg acgcctcgtt cttagttttc ttctcctttttcacaaacaa tgtgcccata 2160 aat 2163 2 653 PRT Plant 2 Met Ala Glu SerGly Asp Phe Asn Gly Gly Gln Pro Pro Pro His Ser 1 5 10 15 Pro Leu ArgThr Thr Ser Ser Gly Ser Ser Ser Ser Asn Asn Arg Gly 20 25 30 Pro Pro ProPro Pro Pro Pro Pro Leu Val Met Val Arg Lys Arg Leu 35 40 45 Ala Ser GluMet Ser Ser Asn Pro Asp Tyr Asn Asn Ser Ser Arg Pro 50 55 60 Pro Arg ArgVal Ser His Leu Leu Asp Ser Asn Tyr Asn Thr Val Thr 65 70 75 80 Pro GlnGln Pro Pro Ser Leu Thr Ala Ala Ala Thr Val Ser Ser Gln 85 90 95 Pro AsnPro Pro Leu Ser Val Cys Gly Phe Ser Gly Leu Pro Val Phe 100 105 110 ProSer Asp Arg Gly Gly Arg Asn Val Met Met Ser Val Gln Pro Met 115 120 125Asp Gln Asp Ser Ser Ser Ser Ser Ala Ser Pro Thr Val Trp Val Asp 130 135140 Ala Ile Ile Arg Asp Leu Ile His Ser Ser Thr Ser Val Ser Ile Pro 145150 155 160 Gln Leu Ile Gln Asn Val Arg Asp Ile Ile Phe Pro Cys Asn ProAsn 165 170 175 Leu Gly Ala Leu Leu Glu Tyr Arg Leu Arg Ser Leu Met LeuLeu Asp 180 185 190 Pro Ser Ser Ser Ser Asp Pro Ser Pro Gln Thr Phe GluPro Leu Tyr 195 200 205 Gln Ile Ser Asn Asn Pro Ser Pro Pro Gln Gln GlnGln Gln His Gln 210 215 220 Gln Gln Gln Gln Gln His Lys Pro Pro Pro ProPro Ile Gln Gln Gln 225 230 235 240 Glu Arg Glu Asn Ser Ser Thr Asp AlaPro Pro Gln Pro Glu Thr Val 245 250 255 Thr Ala Thr Val Pro Ala Val GlnThr Asn Thr Ala Glu Ala Leu Arg 260 265 270 Glu Arg Lys Glu Glu Ile LysArg Gln Lys Gln Asp Glu Glu Gly Leu 275 280 285 His Leu Leu Thr Leu LeuLeu Gln Cys Ala Glu Ala Val Ser Ala Asp 290 295 300 Asn Leu Glu Glu AlaAsn Lys Leu Leu Leu Glu Ile Ser Gln Leu Ser 305 310 315 320 Thr Pro TyrGly Thr Ser Ala Gln Arg Val Ala Ala Tyr Phe Ser Glu 325 330 335 Ala MetSer Ala Arg Leu Leu Asn Ser Cys Leu Gly Ile Tyr Ala Ala 340 345 350 LeuPro Ser Arg Trp Met Pro Gln Thr His Ser Leu Lys Met Val Ser 355 360 365Ala Phe Gln Val Phe Asn Gly Ile Ser Pro Leu Val Lys Phe Ser His 370 375380 Phe Thr Ala Asn Gln Ala Ile Gln Glu Ala Phe Glu Lys Glu Asp Ser 385390 395 400 Val His Ile Ile Asp Leu Asp Ile Met Gln Gly Leu Gln Trp ProGly 405 410 415 Leu Phe His Ile Leu Ala Ser Arg Pro Gly Gly Pro Pro HisVal Arg 420 425 430 Leu Thr Gly Leu Gly Thr Ser Met Glu Ala Leu Gln AlaThr Gly Lys 435 440 445 Arg Leu Ser Asp Phe Thr Asp Lys Leu Gly Leu ProPhe Glu Phe Cys 450 455 460 Pro Leu Ala Glu Lys Val Gly Asn Leu Asp ThrGlu Arg Leu Asn Val 465 470 475 480 Arg Lys Arg Glu Ala Val Ala Val HisTrp Leu Gln His Ser Leu Tyr 485 490 495 Asp Val Thr Gly Ser Asp Ala HisThr Leu Trp Leu Leu Gln Arg Leu 500 505 510 Ala Pro Lys Val Val Thr ValVal Glu Gln Asp Leu Ser His Ala Gly 515 520 525 Ser Phe Leu Gly Arg PheVal Glu Ala Ile His Tyr Tyr Ser Ala Leu 530 535 540 Phe Asp Ser Leu GlyAla Ser Tyr Gly Glu Glu Ser Glu Glu Arg His 545 550 555 560 Val Val GluGln Gln Leu Leu Ser Lys Glu Ile Arg Asn Val Leu Ala 565 570 575 Val GlyGly Pro Ser Arg Ser Gly Glu Val Lys Phe Glu Ser Trp Arg 580 585 590 GluLys Met Gln Gln Cys Gly Phe Lys Gly Ile Ser Leu Ala Gly Asn 595 600 605Ala Ala Thr Gln Ala Thr Leu Leu Leu Gly Met Phe Pro Ser Asp Gly 610 615620 Tyr Thr Leu Val Asp Asp Asn Gly Thr Leu Lys Leu Gly Trp Lys Asp 625630 635 640 Leu Ser Leu Leu Thr Ala Ser Ala Trp Thr Pro Arg Ser 645 6503 23 PRT Plant 3 Pro Ala Val Gln Thr Asn Thr Ala Glu Ala Leu Arg Glu ArgLys Glu 1 5 10 15 Glu Ile Lys Arg Gln Lys Gln 20 4 23 PRT Plant 4 LeuLys Arg Ala Arg Asn Thr Glu Ala Ala Arg Arg Ser Arg Ala Arg 1 5 10 15Lys Leu Gln Arg Met Lys Gln 20 5 23 PRT Plant 5 Arg Arg Leu Ala Gln AsnArg Glu Ala Ala Arg Lys Ser Arg Leu Arg 1 5 10 15 Lys Lys Ala Tyr ValGln Gln 20 6 23 PRT Plant 6 Ile Arg Arg Glu Arg Asn Lys Met Ala Ala AlaLys Cys Arg Asn Arg 1 5 10 15 Arg Arg Glu Leu Thr Asp Thr 20 7 23 PRTPlant 7 Arg Lys Arg Met Arg Asn Arg Ile Ala Ala Ser Lys Cys Arg Lys Arg1 5 10 15 Lys Leu Glu Arg Ile Ala Arg 20 8 23 PRT Plant 8 Val Arg LeuMet Lys Asn Arg Glu Ala Ala Arg Glu Cys Arg Arg Lys 1 5 10 15 Lys LysGlu Tyr Val Lys Cys 20 9 23 PRT Plant 9 Lys Arg Lys Glu Ser Asn Arg GluSer Ala Arg Arg Ser Arg Tyr Arg 1 5 10 15 Lys Ala Ala His Leu Lys Glu 2010 23 PRT Plant 10 Met Arg Gln Ile Arg Asn Arg Asp Ser Ala Met Lys SerArg Glu Arg 1 5 10 15 Lys Lys Ser Tyr Ile Lys Asp 20 11 23 PRT Plant 11Arg Arg Met Val Ser Asn Arg Glu Ser Ala Arg Arg Ser Arg Lys Lys 1 5 1015 Lys Gln Ala His Leu Ala Asp 20 12 43 PRT Plant 12 Ala Phe Glu Lys GluAsp Ser Val His Ile Ile Asp Leu Asp Ile Met 1 5 10 15 Gln Gly Leu GlnTrp Pro Gly Leu Phe His Ile Leu Ala Ser Arg Pro 20 25 30 Gly Gly Pro ProHis Val Arg Leu Thr Gly Leu 35 40 13 43 PRT Plant 13 Ala Val Lys Asn GluSer Phe Val His Ile Ile Asp Phe Gln Ile Ser 1 5 10 15 Gln Gly Gly GlnTrp Val Ser Leu Ile Arg Ala Leu Gly Ala Arg Pro 20 25 30 Gly Gly Pro ProAsn Val Arg Ile Thr Gly Ile 35 40 14 43 PRT Plant 14 Ala Met Glu Gly GluLys Met Val His Val Ile Asp Leu Asp Ala Ser 1 5 10 15 Glu Pro Ala GlnTrp Leu Ala Leu Leu Gln Ala Phe Asn Ser Arg Pro 20 25 30 Glu Gly Pro ProHis Leu Arg Ile Thr Gly Val 35 40 15 29 PRT Plant 15 Ala Ile Lys Gly GluGlu Glu Val His Ile Ile Asp Phe Asp Ile Asn 1 5 10 15 Gln Gly Asn GlnTyr Met Thr Leu Ile Arg Ser Ile Ala 20 25 16 26 PRT Plant VARIANT(1)...(26) Xaa = Any Amino Acid 16 Ile His Val Ile Asp Phe Xaa Leu GlyVal Gly Gly Gln Trp Ala Ser 1 5 10 15 Phe Leu Gln Glu Leu Ala His ArgArg Gly 20 25 17 36 PRT Plant VARIANT (1)...(36) Xaa = Any Amino Acid 17Val His Ile Ile Xaa Phe Xaa Leu Met Gln Gly Leu Gln Trp Pro Ala 1 5 1015 Leu Met Asp Val Phe Ser Ala Arg Lys Gly Gly Pro Pro Lys Leu Arg 20 2530 Ile Thr Gly Ile 35 18 1085 DNA Plant misc_feature (1)...(1085) n =A,T,C or G 18 ggcacgagcc caacgggtcc tgagcttctt acttatatgc atatcttgtatgaagcctgc 60 ccttatttca aattcggtta tgaatctgct aatggagcta tagctgaagctgtgaagaac 120 gaaagttttg tgcacattat cgatttccag atttctcaag gtggtcaatgggtgagtttg 180 atccgtgctc ttggtgctag acctggtgga cctccgaacg ttaggataacgggaattgat 240 gatccgagat catcgtttgc tcgtcaagga ggacttgagt tagttggacaaagacttggg 300 aagctagctg aaatgtgcgg tgttccgttt gagttccatg gagctgctttatgctgcacg 360 gaagtcgaaa tcgagaagct aggagttaga aatggagaag cgctcgcggttaacttcccg 420 cttgttcttc accacatgcc tgatgagagt gtaactgtgg agaatcacagagatagattg 480 ttgagattgg tcaaacactt gtcaccaaac gttgtgactc tggttgagcaagaagcgaat 540 acaaacactg cgccgtttct tccccggttt gtcgagacaa tgaaccattacttggcagtt 600 ttcgaatcaa tagatgtgaa actcgctaga gatcacaagg aaaggatcaatgttgagcag 660 cattgtttgg ctagagaggt tgtgaatctt atagcttgtg aaggtgttgaaagagaagag 720 aggcacgagc cactagggaa atggaggtct cggtttcaca tggcgggatttaaaccgtat 780 cctttgagct cgtatgtgaa cgcaacaatc aaaggattgc ttgagagttattcagagaag 840 tatacacttg aagaaagaga tggagcattg tatttaggat ggaagaatcaacctcttatc 900 acttcttgtg cttggaggta actaataaaa accttgttcg gtttcagaagagattagaaa 960 cttcttttaa agtttgcaga atctgtttgt aaaagtaaaa ctcatgcatgatccgnagga 1020 acaagttgtc aaatgttgta gtagtaagtg atatgttgat gacccaaaaaaaaaaaaaaa 1080 aaaaa 1085 19 306 PRT Plant 19 Gly Thr Ser Pro Thr GlyPro Glu Leu Leu Thr Tyr Met His Ile Leu 1 5 10 15 Tyr Glu Ala Cys ProTyr Phe Lys Phe Gly Tyr Glu Ser Ala Asn Gly 20 25 30 Ala Ile Ala Glu AlaVal Lys Asn Glu Ser Phe Val His Ile Ile Asp 35 40 45 Phe Gln Ile Ser GlnGly Gly Gln Trp Val Ser Leu Ile Arg Ala Leu 50 55 60 Gly Ala Arg Pro GlyGly Pro Pro Asn Val Arg Ile Thr Gly Ile Asp 65 70 75 80 Asp Pro Arg SerSer Phe Ala Arg Gln Gly Gly Leu Glu Leu Val Gly 85 90 95 Gln Arg Leu GlyLys Leu Ala Glu Met Cys Gly Val Pro Phe Glu Phe 100 105 110 His Gly AlaAla Leu Phe Cys Thr Glu Val Glu Ile Glu Lys Leu Gly 115 120 125 Val ArgAsn Gly Glu Ala Leu Ala Val Asn Phe Pro Leu Val Leu His 130 135 140 HisMet Pro Asp Glu Ser Val Thr Val Glu Asn His Arg Asp Arg Leu 145 150 155160 Leu Arg Leu Val Lys His Leu Ser Pro Asn Val Val Thr Leu Val Glu 165170 175 Gln Glu Ala Asn Thr Asn Thr Ala Pro Phe Leu Pro Arg Phe Val Glu180 185 190 Thr Met Asn His Tyr Leu Ala Val Phe Glu Ser Ile Asp Val LysLeu 195 200 205 Ala Arg Asp His Lys Glu Arg Ile Asn Val Glu Gln His CysLeu Ala 210 215 220 Arg Glu Val Glu Asn Leu Ile Ala Cys Glu Gly Val GluArg Glu Glu 225 230 235 240 Arg His Glu Pro Leu Gly Lys Trp Arg Ser ArgPhe His Met Ala Gly 245 250 255 Phe Lys Pro Tyr Pro Leu Ser Ser Tyr ValAsn Ala Thr Ile Lys Gly 260 265 270 Leu Leu Glu Ser Tyr Ser Glu Lys TyrThr Leu Glu Glu Arg Asp Gly 275 280 285 Ala Leu Tyr Leu Gly Trp Lys AsnGln Pro Leu Ile Thr Ser Cys Ala 290 295 300 Trp Arg 305 20 1231 DNAPlant 20 gctatggaag gagagaagat ggttcatgtg attgatctcg atgcttctgagccagctcaa 60 tggcttgctt tgcttcaagc ttttaactct aggcctgaag gtccacctcatttgagaatc 120 actggtgttc atcaccagaa ggaagtgctt gaacaaatgg ctcatagactcattgaggaa 180 gcagagaaac tcgatatccc gtttcagttt aatcccgttg tgagtaggttagactgttta 240 aatgtagaac agttgcgggt taaaacagga gaggccttag ccgttagctcggttcttcaa 300 ttgcatacct tcttggcctc tgatgatgat ctcatgagaa agaactgcgctttacggttt 360 cagaacaacc ctagtggagt tgacttgcag agagttctaa tgatgagccatggctctgca 420 gctgaggcac gtgagaatga tatgagtaac aacaatgggt atagccctagcggtgactcg 480 gcctcatctt tgcctttacc aagttcagga aggactgata gcttcctcaatgctatttgg 540 ggtttgtctc caaaggtcat ggtggtcact gagcaagact cagaccacaacggctccaca 600 ctaatggaga ggctattaga atcactttac acctacgcag cattgtttgattgcttggaa 660 acaaaagttc caagaacgtc tcaagatagg atcaaagtgg agaagatgctcttcggggag 720 gagatcaaga acatcatatc ctgcgaggga tttgagagaa gagaaagacacgagaagctt 780 gagaaatgga gccagaggat cgatttggct ggttttggga atgttcctcttagctattat 840 gcgatgttgc aggctaggag attgcttcaa gggtgcggtt ttgatgggtatagaatcaag 900 gaagagagcg ggtgcgcagt aatttgctgg caagatcgac ctctatactcggtatcagct 960 tggagatgca ggaagtgaat gatatattac agtttgtctt ctattttggttatgagcaga 1020 gtccctttct tttttgtata catggggaca caatcttagt tgttttgtgatggtgacttt 1080 ctgtctcttt atgctatttt ggcttaaatg cttctactgc ctctgcatgtaaagcctttg 1140 tgtgttggtt caatttggtc tggtgtgggt gtaataccaa accaaatccaatttgagctg 1200 aagataacta atttgatgat cggctcgtgc c 1231 21 325 PRT Plant21 Ala Met Glu Gly Glu Lys Met Val His Val Ile Asp Leu Asp Ala Ser 1 510 15 Glu Pro Ala Gln Trp Leu Ala Leu Leu Gln Ala Phe Asn Ser Arg Pro 2025 30 Glu Gly Pro Pro His Leu Arg Ile Thr Gly Val His His Gln Lys Glu 3540 45 Val Leu Glu Gln Met Ala His Arg Leu Ile Glu Glu Ala Glu Lys Leu 5055 60 Asp Ile Pro Phe Gln Phe Asn Pro Val Val Ser Arg Leu Asp Cys Leu 6570 75 80 Asn Val Glu Gln Leu Arg Val Lys Thr Gly Glu Ala Leu Ala Val Ser85 90 95 Ser Val Leu Gln Leu His Thr Phe Leu Ala Ser Asp Asp Asp Leu Met100 105 110 Arg Lys Asn Cys Ala Leu Arg Phe His Asn Asn Pro Ser Gly ValAsp 115 120 125 Leu Gln Arg Val Leu Met Met Ser His Gly Ser Ala Ala GluAla Arg 130 135 140 Glu Asn Asp Met Ser Asn Asn Asn Gly Tyr Ser Pro SerGly Asp Ser 145 150 155 160 Ala Ser Ser Leu Pro Leu Pro Ser Ser Gly ArgThr Asp Ser Phe Leu 165 170 175 Asn Ala Ile Trp Gly Leu Ser Pro Lys ValMet Val Val Thr Glu Gln 180 185 190 Asp Ser Asp His Asn Gly Ser Thr LeuMet Glu Arg Leu Leu Glu Ser 195 200 205 Leu Tyr Thr Tyr Ala Ala Leu PheAsp Cys Leu Glu Thr Lys Val Pro 210 215 220 Arg Thr Ser Gln Asp Arg IleLys Val Glu Lys Met Leu Phe Gly Glu 225 230 235 240 Glu Ile Lys Asn IleIle Ser Cys Glu Gly Phe Glu Arg Arg Glu Arg 245 250 255 His Glu Lys LeuGlu Lys Trp Ser Gln Arg Ile Asp Leu Ala Gly Phe 260 265 270 Gly Asn ValPro Leu Ser Tyr Tyr Ala Met Leu Gln Ala Arg Arg Leu 275 280 285 Leu GlnGly Cys Gly Phe Asp Gly Tyr Arg Ile Lys Glu Glu Ser Gly 290 295 300 CysAla Val Ile Cys Trp Gln Asp Arg Pro Leu Tyr Ser Val Ser Ala 305 310 315320 Trp Arg Cys Arg Lys 325 22 1368 DNA Plant 22 ctttgtcaat ggtaaatgagctgaggcaga tagtttctat ccaaggagac ccttctcaga 60 gaatcgcagc ttacatggtggaaggtctag ctgcaagaat ggccgcttca ggaaaattca 120 tctacagagc attgaaatgcaaagagcctc cttcggatga gaggcttgca gctatgcaag 180 tcctgtttga agtctgcccttgtttcaagt tcgggttttt agcagctaat ggtgcgatac 240 ttgaagcaat caaaggtgaagaagaagttc acataatcga tttcgatata aaccaaggga 300 accaatacat gacactgatacgaagcattg ctgagttgcc tggtaaacga cctcgcctga 360 ggttaacagg aattgatgaccctgaatcag tccaacgctc cattggaggg ctaagaatca 420 tcggtctaag actcgagcaactcgcagagg ataatggagt atccttcaaa ttcaaagcaa 480 tgccttcaaa gacttcgattgtctctccat caacactcgg ttgcaaacca ggagaaacct 540 taatagtgaa ctttgcattccaacttcacc acatgcctga cgagagtgtc acaacagtaa 600 accagcggga cgagctacttcacatggtca aaagcttaaa cccaaagctt gtcacggtcg 660 ttgaacaaga cgtgaacacaaacacttcac cgttctttcc cagattcata gaggcttacg 720 aatactactc agcagttttcgagtctctag acatgacact tccaagagaa agccaagaga 780 ggatgaatgt agaaagacagtgtctcgcta gagacatagt caacattgtt gcttgcgaag 840 gagaagaacg gatagagagatacgaggctg cgggaaaatg gagagcaagg atgatgatgg 900 ctggattcaa tccaaaaccaatgagtgcta aagtaaccaa caatatacaa aacctgataa 960 agcaacaata ttgcaataagtacaagctta aagaagaaat gggtgagctc catttttgct 1020 gggaggagaa aagcttaatcgttgcttcag cttggaggta agataagtga caagagcata 1080 tagtctttat gtttcataaaacataattat gtttttactg taatcttggg ttattgtgta 1140 actggttaaa tcatctccatgtattattac cagaggttag gggtgatcac aggtactaaa 1200 agctaatcta acacttatggaagaattttt ctttcttttt tttccctatt atataaaaat 1260 aattagagtt ttggttctaaacctatttgc taagtgtgaa tgagtcttta catgttcata 1320 tttcagttca aatggttaaatttgttaagg ttctcactta aaaaaaaa 1368 23 351 PRT Plant 23 Leu Ser Met ValAsn Glu Leu Arg Gln Ile Val Ser Ile Gln Gly Asp 1 5 10 15 Pro Ser GlnArg Ile Ala Ala Tyr Met Val Glu Gly Leu Ala Ala Arg 20 25 30 Met Ala AlaSer Gly Lys Phe Ile Tyr Arg Ala Leu Lys Cys Lys Glu 35 40 45 Pro Pro SerAsp Glu Arg Leu Ala Ala Met Gln Val Leu Phe Glu Val 50 55 60 Cys Pro CysPhe Lys Phe Gly Phe Leu Ala Ala Asn Gly Ala Ile Leu 65 70 75 80 Glu AlaIle Lys Gly Glu Glu Glu Val His Ile Ile Asp Phe Asp Ile 85 90 95 Asn GlnGly Asn Gln Tyr Met Thr Leu Ile Arg Ser Ile Ala Glu Leu 100 105 110 ProGly Lys Arg Pro Arg Leu Arg Leu Thr Gly Ile Asp Asp Pro Glu 115 120 125Ser Val Gln Arg Ser Ile Gly Gly Leu Arg Ile Ile Asn Leu Arg Leu 130 135140 Glu Gln Leu Ala Glu Asp Asn Gly Val Ser Phe Lys Phe Lys Ala Met 145150 155 160 Pro Ser Lys Thr Ser Ile Val Ser Pro Ser Thr Leu Gly Cys LysPro 165 170 175 Gly Glu Thr Leu Ile Val Asn Phe Ala Phe Gln Leu His HisMet Pro 180 185 190 Asp Glu Ser Val Thr Thr Val Asn Gln Arg Asp Glu LeuLeu His Met 195 200 205 Val Lys Ser Leu Asn Pro Leu Val Thr Val Val GluGln Asp Val Asn 210 215 220 Thr Asn Thr Ser Pro Phe Phe Pro Arg Phe IleGlu Ala Tyr Glu Tyr 225 230 235 240 Tyr Ser Ala Val Phe Glu Ser Leu AspMet Thr Leu Pro Arg Glu Ser 245 250 255 Gln Glu Arg Met Asn Val Glu ArgGln Cys Leu Ala Arg Asp Ile Val 260 265 270 Asn Ile Val Ala Cys Glu GlyGlu Glu Arg Ile Glu Arg Tyr Glu Ala 275 280 285 Ala Gly Lys Trp Arg AlaArg Met Met Met Ala Gly Phe Asn Pro Lys 290 295 300 Pro Met Ser Ala LysVal Thr Asn Asn Ile Gln Asn Leu Ile Lys Gln 305 310 315 320 Gln Tyr CysAsn Lys Tyr Lys Leu Lys Glu Glu Met Gly Glu Leu His 325 330 335 Phe CysTrp Glu Glu Lys Ser Leu Ile Val Ala Ser Ala Trp Arg 340 345 350 24 100DNA Plant 24 ccaggaggcg ttcgagcggg aggagcgtgt gcacatcatc gacctcgacatcatgcaggg 60 gctgcagtgg ccgggcctcc tccacatcct tgcctcccgc 100 25 33 PRTPlant 25 Gln Glu Ala Phe Glu Arg Glu Glu Arg Val His Ile Ile Asp Leu Asp1 5 10 15 Ile Met Gln Gly Leu Gln Trp Pro Gly Leu Phe His Ile Leu AlaSer 20 25 30 Arg 26 1094 DNA Plant 26 ccacgcgtcc gtcaaaggat acaaccatgtacacataatt gacttttccc tgatgcaagg 60 tctccagtgg ccggcactca tggatgtcttctccgcccgt gagggtgggc caccaaagct 120 ccgaatcaca ggcattggcc cgaacccaataggtggccgt gacgagctcc atgaagtggg 180 aattcgcctc gccaagtatg cacactcggtgggtatcgac ttcactttcc agggagtctg 240 tgtcgatcaa cttgataggt tgtgcgactggatgcttctc aaaccaatca aaggagaggc 300 agttgccata aactccatcc tacaactccatcgcctcctc gttgacccag atgcaaaccc 360 agtggtgccc gcaccaatag atatcctcctcaaattggtc atcaagataa accccatgat 420 cttcacggtg gttgagcatg aggcagatcacaacagacca ccactactag agaggttcac 480 taatgccctc ttccactatg cgaccatgtttgactctttg gaggccatgc atcgttgtac 540 cagtggtaga gacatcaccg actcactcacagaggtgtac cttcgaggtg agatttttga 600 cattgtctgc ggcgagggca gtgcacgcaccgaacgtcat gagttgtttg gtcactggag 660 ggagaggctc acctatgctg ggctaactcaagtgtggttc gaccccgatg aggttgacac 720 gctaaaagac cagttgatcc atgtgacatccttatctggc tctgggttca acatcctagt 780 gtgtgatggc agccttgcac tagcgtggcataatcgcccg ttatatgtgg caacagcttg 840 gtgtgtgaca ggaggaaatg ctgccagttccatggttggc aacatctgta agggtacaaa 900 tgatagtaga agaaaggaaa accgtaatggacccatggag tagcaggaag aataaccatg 960 tcatgagcaa atcgatcaag taataaaatgcactgatgac atgcatggtg atctaaagtt 1020 tttttgcgtg aatgtgcaat gacgaattgttcaatttgaa taacctaatc atgagactca 1080 aaaaaaaaaa aaaa 1094 27 313 PRTPlant 27 His Ala Ser Val Lys Gly Tyr Asn His Val His Ile Ile Asp Phe Ser1 5 10 15 Leu Met Gln Gly Leu Gln Trp Pro Ala Leu Met Asp Val Phe SerAla 20 25 30 Arg Glu Gly Gly Pro Pro Lys Leu Arg Ile Thr Gly Ile Gly ProAsn 35 40 45 Pro Ile Gly Gly Arg Asp Glu Leu His Glu Val Gly Ile Arg LeuAla 50 55 60 Lys Tyr Ala His Ser Val Gly Ile Asp Phe Thr Phe Gln Gly ValCys 65 70 75 80 Val Asp Gln Leu Asp Arg Leu Cys Asp Trp Met Leu Leu LysPro Ile 85 90 95 Lys Gly Glu Ala Val Ala Ile Asn Ser Ile Leu Gln Leu HisArg Leu 100 105 110 Leu Val Asp Pro Asp Ala Asn Pro Val Val Pro Ala ProIle Asp Ile 115 120 125 Leu Leu Lys Leu Val Ile Lys Ile Asn Pro Met IlePhe Thr Val Val 130 135 140 Glu His Glu Ala Asp His Asn Arg Pro Pro LeuLeu Glu Arg Phe Thr 145 150 155 160 Asn Ala Leu Phe His Tyr Ala Thr MetPhe Asp Ser Leu Glu Ala Met 165 170 175 His Arg Cys Thr Ser Gly Arg AspIle Thr Asp Ser Leu Thr Glu Val 180 185 190 Tyr Leu Arg Gly Glu Ile PheAsp Ile Val Cys Gly Glu Gly Ser Ala 195 200 205 Arg Thr Glu Arg His GluLeu Phe Gly His Trp Arg Glu Arg Leu Thr 210 215 220 Tyr Ala Gly Leu ThrGln Val Trp Phe Asp Pro Asp Glu Val Asp Thr 225 230 235 240 Leu Lys AspGln Leu Ile His Val Thr Ser Leu Ser Gly Ser Gly Phe 245 250 255 Asn IleLeu Val Cys Asp Gly Ser Leu Ala Leu Ala Trp His Asn Arg 260 265 270 ProLeu Tyr Val Ala Thr Ala Trp Cys Val Thr Gly Gly Asn Ala Ala 275 280 285Ser Ser Met Val Gly Asn Ile Cys Lys Gly Thr Asn Asp Ser Arg Arg 290 295300 Lys Glu Asn Arg Asn Gly Pro Met Glu 305 310 28 611 DNA Plant 28cccaacttgg gaagcccttc ctccgctccg cctcctacct caaggaggcc ctcctcctcg 60cactcgccga cagccaccat ggctcctccg gcgtcacctc gccgctcgac gttgccctca 120agcttgcagc atacaagtct ttctctgacc tgtcacctgt gctccagttc actaacttta 180ccgcaacaag gcgcttcttg atgagattgg tggcatggca acttcctgca tccatgtcat 240tgactttgat ctcggtgttg gtggtcagtg ggcttccttc ttgcaggagc ttgcccaccg 300ccggggagct ggaggtatgg ccttgccgtt gttgaagctc acggctttca tgtcgactgc 360ttctcaccat ccactggagc tgcaccttac ccaggataac ctctctcagt ttgccgcaga 420gctcagaatt cctttcgaat tcaatgccgt cagtcttgat gcattcaatc ctgcggaatc 480tatttcttcc tctggtgatg aagttgttgc tgttagcctc cctgttggct gctctgctcg 540tgcaccaccg ctgccagcga ttcttcggtt ggtgaaacag ctttgtccta aggttgtcgt 600ggctattgat c 611 29 502 DNA Plant 29 tttttttttt tttttttttt tttttttttttacagagcaa cagcagtata atattaattc 60 tgtaccacac aaccatttga taggttaaattaccctctag tctctactca taagcagtgt 120 ttccaatgag atgatcatgg ctaattgagcagagcatggc aacaacctaa agcaacatca 180 ttagctatag agactgacac caatattcctaaatccacta ggctagctaa taagctgcaa 240 cgaaaagcaa tatgaagagt tcaacagctcaagacaacaa tttcatttgc aacatttaat 300 tgcaagaata aatggacatt actggagtggtcgatgcttg caaacggtgg tggaaccttg 360 gtggagtgaa gcttatggct gatcagcaccgccaagatga tatggataca agctccccac 420 gctgccagta gagcgtaaga gcagctccgcgtttctccac atggaatcct cggacctgca 480 cccgcttcag gaggcagtct gc 502 30 298PRT Plant 30 Pro Gln Gln Gln Gln Gln His Gln Gln Gln Gln Gln Gln His LysPro 1 5 10 15 Pro Pro Pro Pro Ile Gln Gln Gln Glu Arg Glu Asn Ser SerThr Asp 20 25 30 Ala Pro Pro Gln Pro Glu Thr Val Thr Ala Thr Val Pro AlaVal Gln 35 40 45 Thr Asn Thr Ala Glu Ala Leu Arg Glu Arg Lys Glu Glu IleLys Arg 50 55 60 Gln Lys Gln Asp Glu Glu Gly Leu His Leu Leu Thr Leu LeuLeu Gln 65 70 75 80 Cys Ala Glu Ala Val Ser Ala Asp Asn Leu Glu Glu AlaAsn Lys Leu 85 90 95 Leu Leu Glu Ile Ser Gln Leu Ser Thr Pro Tyr Gly ThrSer Ala Gln 100 105 110 Arg Val Ala Ala Tyr Phe Ser Glu Ala Met Ser AlaArg Leu Leu Asn 115 120 125 Ser Cys Leu Gly Ile Tyr Ala Ala Leu Pro SerArg Trp Met Pro Gln 130 135 140 Thr His Ser Leu Lys Met Val Ser Ala PheGln Val Phe Asn Gly Ile 145 150 155 160 Ser Pro Leu Val Lys Phe Ser HisPhe Thr Ala Asn Gln Ala Ile Gln 165 170 175 Glu Ala Phe Glu Lys Glu AspSer Val His Ile Ile Asp Leu Asp Ile 180 185 190 Met Gln Gly Leu Gln TrpPro Gly Leu Phe His Ile Leu Ala Ser Arg 195 200 205 Pro Gly Gly Pro ProHis Val Arg Leu Thr Gly Leu Gly Thr Ser Met 210 215 220 Glu Ala Leu GlnAla Thr Gly Lys Arg Leu Ser Asp Phe Thr Asp Lys 225 230 235 240 Leu GlyLeu Pro Phe Glu Phe Cys Pro Leu Ala Glu Lys Val Gly Asn 245 250 255 AspLeu Thr Glu Arg Leu Asn Val Arg Lys Arg Glu Ala Ala Val His 260 265 270Trp Leu Gln His Ser Leu Tyr Asp Val Thr Gly Ser Asp Ala His Thr 275 280285 Leu Trp Leu Leu Gln Arg Leu Ala Pro Lys 290 295 31 307 PRT Plant 31Gly Thr Ser Pro Thr Gly Pro Glu Leu Leu Thr Tyr Met His Ile Leu 1 5 1015 Tyr Glu Ala Cys Pro Tyr Phe Lys Phe Gly Tyr Glu Ser Ala Asn Gly 20 2530 Ala Ile Ala Glu Ala Val Lys Asn Glu Ser Phe Val His Ile Ile Asp 35 4045 Phe Gln Ile Ser Gln Gly Gly Gln Trp Val Ser Leu Ile Arg Ala Leu 50 5560 Gly Ala Arg Pro Gly Gly Pro Pro Asn Val Arg Ile Thr Gly Ile Asp 65 7075 80 Asp Pro Arg Ser Ser Phe Ala Arg Gln Gly Gly Leu Glu Leu Val Gly 8590 95 Gln Arg Leu Gly Lys Leu Ala Glu Met Cys Gly Val Pro Phe Glu Phe100 105 110 His Gly Ala Ala Leu Cys Cys Thr Glu Val Glu Ile Glu Lys LeuGly 115 120 125 Val Arg Asn Gly Glu Ala Leu Ala Val Asn Phe Pro Leu ValLeu His 130 135 140 His Met Pro Asp Glu Ser Val Thr Val Glu Asn His ArgAsp Arg Leu 145 150 155 160 Leu Arg Leu Val Lys His Leu Ser Pro Asn ValVal Thr Leu Val Glu 165 170 175 Gln Glu Ala Asn Thr Asn Thr Ala Pro PheLeu Pro Arg Phe Val Glu 180 185 190 Thr Met Asn His Tyr Leu Ala Val PheGlu Ser Ile Asp Val Lys Leu 195 200 205 Ala Arg Asp His Lys Glu Arg IleAsn Val Glu Gln His Cys Leu Ala 210 215 220 Arg Glu Val Val Asn Leu IleAla Cys Glu Gly Val Glu Arg Glu Glu 225 230 235 240 Arg His Glu Pro LeuGly Lys Trp Arg Ser Arg Phe His Met Ala Gly 245 250 255 Phe Lys Pro TyrPro Leu Ser Ser Tyr Val Asn Ala Thr Ile Lys Gly 260 265 270 Leu Leu GluSer Tyr Ser Glu Lys Tyr Thr Leu Glu Glu Arg Asp Gly 275 280 285 Ala LeuTyr Leu Gly Trp Lys Asn Gln Pro Leu Ile Thr Ser Cys Ala 290 295 300 TrpArg Xaa 305 32 353 PRT Plant 32 Leu Ser Met Val Asn Glu Leu Arg Gln IleVal Ser Ile Gln Gly Asp 1 5 10 15 Pro Ser Gln Arg Ile Ala Ala Tyr MetVal Glu Gly Leu Ala Ala Arg 20 25 30 Met Ala Ala Ser Gly Lys Phe Ile TyrArg Ala Leu Lys Cys Lys Glu 35 40 45 Pro Pro Ser Asp Glu Arg Leu Ala AlaMet Gln Val Leu Phe Glu Val 50 55 60 Cys Pro Cys Phe Lys Phe Gly Phe LeuAla Ala Asn Gly Ala Ile Leu 65 70 75 80 Glu Ala Ile Lys Gly Glu Glu GluVal His Ile Ile Asp Phe Asp Ile 85 90 95 Asn Gln Gly Asn Gln Tyr Met ThrLeu Ile Arg Ser Ile Ala Glu Leu 100 105 110 Pro Gly Lys Arg Pro Arg LeuArg Leu Thr Gly Ile Asp Asp Pro Glu 115 120 125 Ser Val Gln Arg Ser IleGly Gly Leu Arg Ile Ile Gly Leu Arg Leu 130 135 140 Glu Gln Leu Ala GluAsp Asn Gly Val Ser Phe Lys Phe Lys Ala Met 145 150 155 160 Pro Ser LysThr Ser Ile Val Ser Pro Ser Thr Leu Gly Cys Lys Pro 165 170 175 Gly GluThr Leu Ile Val Asn Phe Ala Phe Gln Leu His His Met Pro 180 185 190 AspGlu Ser Val Thr Thr Val Asn Gln Arg Asp Glu Leu Leu His Met 195 200 205Val Lys Ser Leu Asn Pro Lys Leu Val Thr Val Val Glu Gln Asp Val 210 215220 Asn Thr Asn Thr Ser Pro Phe Phe Pro Arg Phe Ile Glu Ala Tyr Glu 225230 235 240 Tyr Tyr Ser Ala Val Phe Glu Ser Leu Asp Met Thr Leu Pro ArgGlu 245 250 255 Ser Gln Glu Arg Met Asn Val Glu Arg Gln Cys Leu Ala ArgAsp Ile 260 265 270 Val Asn Ile Val Ala Cys Glu Gly Glu Glu Arg Ile GluArg Tyr Glu 275 280 285 Ala Ala Gly Lys Trp Arg Ala Arg Met Met Met AlaGly Phe Asn Pro 290 295 300 Lys Pro Met Ser Ala Lys Val Thr Asn Asn IleGln Asn Leu Ile Lys 305 310 315 320 Gln Gln Tyr Cys Asn Lys Tyr Lys LeuLys Glu Glu Met Gly Glu Leu 325 330 335 His Phe Cys Trp Glu Glu Lys SerLeu Ile Val Ala Ser Ala Trp Arg 340 345 350 Xaa 33 326 PRT Plant 33 AlaMet Glu Gly Glu Lys Met Val His Val Ile Asp Leu Asp Ala Ser 1 5 10 15Glu Pro Ala Gln Trp Leu Ala Leu Leu Gln Ala Phe Asn Ser Arg Pro 20 25 30Glu Gly Pro Pro His Leu Arg Ile Thr Gly Val His His Gln Lys Glu 35 40 45Val Leu Glu Gln Met Ala His Arg Leu Ile Glu Glu Ala Glu Lys Leu 50 55 60Asp Ile Pro Phe Gln Phe Asn Pro Val Val Ser Arg Leu Asp Cys Leu 65 70 7580 Asn Val Glu Gln Leu Arg Val Lys Thr Gly Glu Ala Leu Ala Val Ser 85 9095 Ser Val Leu Gln Leu His Thr Phe Leu Ala Ser Asp Asp Asp Leu Met 100105 110 Arg Lys Asn Cys Ala Leu Arg Phe Gln Asn Asn Pro Ser Gly Val Asp115 120 125 Leu Gln Arg Val Leu Met Met Ser His Gly Ser Ala Ala Glu AlaArg 130 135 140 Glu Asn Asp Met Ser Asn Asn Asn Gly Tyr Ser Pro Ser GlyAsp Ser 145 150 155 160 Ala Ser Ser Leu Pro Leu Pro Ser Ser Gly Arg ThrAsp Ser Phe Leu 165 170 175 Asn Ala Ile Trp Gly Leu Ser Pro Lys Val MetVal Val Thr Glu Gln 180 185 190 Asp Ser Asp His Asn Gly Ser Thr Leu MetGlu Arg Leu Leu Glu Ser 195 200 205 Leu Tyr Thr Tyr Ala Ala Leu Phe AspCys Leu Glu Thr Lys Val Pro 210 215 220 Arg Thr Ser Gln Asp Arg Ile LysVal Glu Lys Met Leu Phe Gly Glu 225 230 235 240 Glu Ile Lys Asn Ile IleSer Cys Glu Gly Phe Glu Arg Arg Glu Arg 245 250 255 His Glu Lys Leu GluLys Trp Ser Gln Arg Ile Asp Leu Ala Gly Phe 260 265 270 Gly Asn Val ProLeu Ser Tyr Tyr Ala Met Leu Gln Ala Arg Arg Leu 275 280 285 Leu Gln GlyCys Gly Phe Asp Gly Tyr Arg Ile Lys Glu Glu Ser Gly 290 295 300 Cys AlaVal Ile Cys Trp Gln Asp Arg Pro Leu Tyr Ser Val Ser Ala 305 310 315 320Trp Arg Cys Arg Lys Xaa 325 34 277 PRT Plant VARIANT (1)...(277) Xaa =Any Amino Acid 34 Asn Lys Arg Leu Lys Ser Cys Ser Ser Pro Asp Ser MetVal Thr Ser 1 5 10 15 Thr Ser Thr Gly Thr Gln Ile Gly Gly Val Ile GlyThr Thr Val Thr 20 25 30 Thr Thr Thr Thr Thr Thr Thr Ala Ala Ala Glu SerThr Arg Ser Val 35 40 45 Ile Leu Val Asp Ser Gln Glu Asn Gly Val Arg LeuVal His Ala Leu 50 55 60 Met Ala Cys Ala Glu Ala Ile Gln Gln Asn Asn LeuThr Leu Ala Glu 65 70 75 80 Ala Leu Val Lys Gln Ile Gly Cys Leu Ala ValSer Gln Ala Gly Ala 85 90 95 Met Arg Lys Val Ala Thr Tyr Phe Ala Glu AlaLeu Ala Arg Arg Ile 100 105 110 Tyr Arg Leu Ser Pro Pro Gln Asn Gln IleAsp His Cys Leu Ser Asp 115 120 125 Thr Leu Gln Met His Phe Tyr Glu ThrCys Pro Tyr Leu Lys Phe Ala 130 135 140 His Phe Thr Ala Asn Gln Ala IleLeu Glu Ala Phe Glu Gly Lys Lys 145 150 155 160 Arg Val His Val Ile AspPhe Ser Met Asn Gln Gly Leu Gln Trp Pro 165 170 175 Ala Leu Met Gln AlaLeu Ala Leu Arg Glu Gly Gly Pro Pro Thr Phe 180 185 190 Arg Leu Thr GlyIle Gly Pro Pro Ala Pro Asp Asn Ser Asp His Leu 195 200 205 His Glu ValGly Cys Lys Leu Ala Gln Leu Ala Glu Ala Ile His Val 210 215 220 Glu PheGlu Tyr Arg Gly Phe Val Ala Asn Ser Leu Ala Asp Leu Asp 225 230 235 240Ala Ser Met Leu Glu Leu Arg Pro Ser Asp Thr Glu Ala Val Ala Val 245 250255 Asn Ser Val Phe Glu Leu His Lys Leu Leu Gly Arg Xaa Gly Gly Ile 260265 270 Glu Lys Val Leu Gly 275 35 262 PRT Plant 35 Gly Gly Gly Gly AspThr Tyr Thr Thr Asn Lys Arg Leu Lys Cys Ser 1 5 10 15 Asn Gly Val ValGlu Thr Thr Thr Ala Thr Ala Glu Ser Thr Arg His 20 25 30 Val Val Leu ValAsp Ser Gln Glu Asn Gly Val Arg Leu Val His Ala 35 40 45 Leu Leu Ala CysAla Glu Ala Val Gln Lys Glu Asn Leu Thr Val Ala 50 55 60 Glu Ala Leu ValLys Gln Ile Gly Phe Leu Ala Val Ser Gln Ile Gly 65 70 75 80 Ala Met ArgGln Val Ala Thr Tyr Phe Ala Glu Ala Leu Ala Arg Arg 85 90 95 Ile Tyr ArgLeu Ser Pro Ser Gln Ser Pro Ile Asp His Ser Leu Ser 100 105 110 Asp ThrLeu Gln Met His Phe Tyr Glu Thr Cys Pro Tyr Leu Lys Phe 115 120 125 AlaHis Phe Thr Ala Asn Gln Ala Ile Leu Glu Ala Phe Gln Gly Lys 130 135 140Lys Arg Val His Val Ile Asp Phe Ser Met Ser Gln Gly Leu Gln Trp 145 150155 160 Pro Ala Leu Met Gln Ala Leu Ala Leu Arg Pro Gly Gly Pro Pro Val165 170 175 Phe Arg Leu Thr Gly Ile Gly Pro Pro Ala Pro Asp Asn Phe AspTyr 180 185 190 Leu His Glu Val Gly Cys Lys Leu Ala His Leu Ala Glu AlaIle His 195 200 205 Val Glu Phe Glu Tyr Arg Gly Phe Val Ala Asn Thr LeuAla Asp Leu 210 215 220 Asp Ala Ser Met Leu Glu Leu Arg Pro Ser Glu IleGlu Ser Val Ala 225 230 235 240 Val Asn Ser Val Phe Glu Leu His Lys LeuLeu Gly Arg Pro Gly Ala 245 250 255 Ile Asp Lys Val Leu Gly 260 36 203PRT Plant 36 Gln Leu Gly Lys Pro Phe Leu Arg Ser Ala Ser Tyr Leu Lys GluAla 1 5 10 15 Leu Leu Leu Ala Leu Ala Asp Ser His His Gly Ser Ser GlyVal Thr 20 25 30 Ser Pro Leu Asp Val Ala Leu Lys Leu Ala Ala Tyr Lys SerPhe Ser 35 40 45 Asp Leu Ser Pro Val Leu Gln Phe Thr Asn Phe Thr Ala AsnLys Ala 50 55 60 Leu Leu Asp Glu Ile Gly Gly Met Ala Thr Ser Cys Ile HisVal Ile 65 70 75 80 Asp Phe Asn Leu Gly Val Gly Gly Gln Trp Ala Ser PheLeu Gln Glu 85 90 95 Leu Ala His Arg Arg Gly Ala Gly Gly Met Ala Leu ProLeu Leu Lys 100 105 110 Leu Thr Ala Phe Met Ser Thr Ala Ser His His ProLeu Glu Leu His 115 120 125 Leu Thr Gln Asp Asn Leu Ser Gln Phe Ala AlaGlu Leu Arg Ile Pro 130 135 140 Phe Glu Phe Asn Ala Val Ser Leu Asp AlaPhe Asn Pro Ala Glu Ser 145 150 155 160 Ile Ser Ser Ser Gly Asp Glu ValVal Ala Val Ser Leu Pro Val Gly 165 170 175 Cys Ser Ala Arg Ala Pro ProLeu Pro Ala Ile Leu Arg Leu Val Lys 180 185 190 Gln Leu Cys Pro Lys ValVal Val Ala Ile Asp 195 200 37 131 PRT Plant 37 His Ala Ser Val Lys GlyTyr Asn His Val His Ile Ile Asp Phe Ser 1 5 10 15 Leu Met Gln Gly LeuGln Trp Pro Ala Leu Met Asp Val Phe Ser Ala 20 25 30 Arg Glu Gly Gly ProPro Lys Leu Arg Ile Thr Gly Ile Gly Pro Asn 35 40 45 Pro Ile Gly Gly ArgAsp Glu Leu His Glu Val Gly Ile Arg Leu Ala 50 55 60 Lys Tyr Ala His SerVal Gly Ile Asp Phe Thr Phe Gln Gly Val Cys 65 70 75 80 Val Asp Gln LeuAsp Arg Leu Cys Asp Trp Met Leu Leu Lys Pro Ile 85 90 95 Lys Gly Glu AlaVal Ala Ile Asn Ser Ile Leu Gln Leu His Arg Leu 100 105 110 Leu Val AspPro Asp Ala Asn Pro Val Val Pro Ala Pro Ile Asp Ile 115 120 125 Leu LeuLys 130 38 33 PRT Plant 38 Gln Glu Ala Phe Glu Arg Glu Glu Arg Val HisIle Ile Asp Leu Asp 1 5 10 15 Ile Met Gln Gly Leu Gln Trp Pro Gly LeuPhe His Ile Leu Ala Ser 20 25 30 Arg 39 29 PRT Plant VARIANT (1)...(29)Xaa = Any Amino Acid 39 Phe Ala Gly Cys Arg Arg Val His Val Val Asp PheGly Ile Lys Gln 1 5 10 15 Gly Met Gln Trp Pro Ala Leu Leu Xaa Asp LeuAla Leu 20 25 40 73 PRT Plant VARIANT (1)...(73) Xaa = Any Amino Acid 40Gly Arg Asn Gly Arg Thr Leu Trp Leu Gly Glu Gly His Ile Asp Leu 1 5 1015 Trp Pro Leu Gln Gly Leu Leu Ser Gln Gly Leu Gln Arg Ala Leu Cys 20 2530 Ala Arg Pro Leu Gly Ala Pro His Val Phe Leu Pro Gly Leu His Thr 35 4045 Leu Ser Leu Gly Leu Gln Xaa Arg His Leu Leu Val His Met Met Ala 50 5560 Leu Ser Tyr Ser Tyr Gly Arg Xaa Pro 65 70 41 59 PRT Plant 41 Thr SerAsp Ser Ala Ser Ser Phe Asn Ile Pro Thr Ser Ala Gln Asn 1 5 10 15 HisTyr Ala Thr Gly Ser Phe Ser Thr Asn Ser Arg Thr Thr Asn Val 20 25 30 AlaThr Ala Thr Thr Asn Ser Ala Thr Ala His Trp Val Ala Thr Asp 35 40 45 AlaGlu His Thr Asp Thr Ile Ile Ala Gln Pro 50 55 42 110 PRT Plant VARIANT(1)...(110) Xaa = Any Amino Acid 42 Arg Xaa Phe Asp Ser Leu Glu His AspAla Ser Lys Gly Glu Pro Arg 1 5 10 15 Glu Asp Glu Arg Gly Arg Xaa CysLeu Ala Arg Asn Ile Val Asn Ile 20 25 30 Val Xaa Cys Lys Xaa Glu Glu ArgIle Glu Arg Tyr Glu Val Thr Gly 35 40 45 Lys Trp Arg Ala Arg Met Met MetAla Gly Phe Ser Pro Arg Pro Met 50 55 60 Ser Gly Arg Val Thr Ser Asn IleGlu Ser Leu Ile Lys Arg Asp Tyr 65 70 75 80 Cys Ser Lys Tyr Lys Val LysGlu Glu Met Gly Glu Leu His Phe Ser 85 90 95 Trp Glu Glu Lys Ser Leu IleVal Ala Ser Ala Trp Ser Xaa 100 105 110 43 137 PRT Plant VARIANT(1)...(137) Xaa = Any Amino Acid 43 Asn Gly Ser Tyr Asn Ala Pro Phe PheVal Thr Arg Phe Arg Glu Ala 1 5 10 15 Leu Phe His Tyr Ser Ala Ile PheAsp Met Leu Glu Thr Asn Ile Pro 20 25 30 Lys Asp Asn Glu Gln Arg Leu LeuIle Glu Ser Ala Leu Phe Ser Arg 35 40 45 Glu Xaa Asn Val Ile Ser Cys GluGly Leu Glu Arg Met Glu Arg Pro 50 55 60 Glu Thr Tyr Lys Gln Trp Gln ValArg Asn Gln Arg Val Gly Phe Lys 65 70 75 80 Gln Leu Pro Leu Asn Gln AspMet Met Lys Arg Ala Arg Xaa Glu Gly 85 90 95 Gln Val Leu Pro Thr Arg ThrPhe Ile Ile Asp Glu Asp Asn Arg Trp 100 105 110 Leu Leu Gln Gly Trp LysGly Arg Ile Leu Phe Ala Leu Ser Thr Trp 115 120 125 Lys Pro Asp Asn ArgSer Ser Ser Xaa 130 135 44 41 PRT Plant 44 Asn Gly Gly Ala Phe Ala ProSer Thr Trp Thr Ala Arg Ser Leu Asn 1 5 10 15 Gly Gly Ala Phe Ala ProSer Thr Trp Thr Ala Arg Ser Leu Pro Val 20 25 30 Pro Ser Ser Pro Ser ThrAsp Ser Phe 35 40 45 1279 DNA Plant 45 gcggctatct tctacggcca ccaccaccatacacctccgc cggcaaagcg gctcaaccct 60 ggtcccgtgg ggataacaga gcagctggttaaggcagcag aggtcataga gagcgacacg 120 tgtctagctc aggggatatt ggcgcggctcaatcaacagc tctcttctcc cgtcgggaag 180 ccattagaaa gagcagcttt ttacttcaaagaagctctca ataatctcct tcacaacgtc 240 tcccaaaccc taaaccctta ttccctcatcttcaagatcg ctgcttacaa atccttctca 300 gagatctctc ccgttcttca gttcgccaactttacctcca accaagccct cttagagtcc 360 ttccatggct tccaccgtct ccacatcatcgacttcgata tcggctacgg tggccaatgg 420 gcttccctca tgcaagagct tgttctccgcgacaacgccg ctcctctctc cctcaagatc 480 accgttttcg cttctccggc gaaccacgaccagctcgaac ttggcttcac tcaagacaac 540 ctcaagcact tcgcctctga gatcaacatctcccttgaca tccaagtttt gagcttagac 600 ctcctcggct ccatctcgtg gcctaactcgtcggagaaag aagctgtcgc cgttaacatc 660 tccgccgcgt ccttctcgca cctccctttggtcctccgtt tcgtgaagca tctatctccg 720 acgatcatcg tctgctccga cagaggatgcgagaggacgg atctgccctt ctctcaacag 780 ctcgcccact cgctgcactc acacaccgctctcttcgaat ccctcgacgc cgtcaacgcc 840 aacctcgacg caatgcagaa gatcgagaggtttcttatac agccggagat agagaagctg 900 gtgttggatc gtagccgtcc gatagaaaggccgatgatga cgtggcaagc gatgtttcta 960 cagatgggtt tctcaccggt gacgcacagtaacttcacgg agtctcaagc cgagtgttta 1020 gtccaacgga cgccagtgag aggctttcacgtcgagaaga aacataactc acttctccta 1080 tgttggcaaa ggacagaact cgtcggagtttcagcatgga gatgtcgctc ctcctgattt 1140 ccaccggagt ttcaattatt aaaaaaatattttccttaat tcaatttatc ttaaatgaca 1200 aatttttagt ttctgatttt attttgctcagtgcgatgga tttttaaatt taagtttcac 1260 acaaatatat aaatttttg 1279 46 379PRT Plant 46 Ala Ala Ile Phe Tyr Gly His His His His Thr Pro Pro Pro AlaLys 1 5 10 15 Arg Leu Asn Pro Gly Pro Val Gly Ile Thr Glu Gln Leu ValLys Ala 20 25 30 Ala Glu Val Ile Glu Ser Asp Thr Cys Leu Ala Gln Gly IleLeu Ala 35 40 45 Arg Leu Asn Gln Gln Leu Ser Ser Pro Val Gly Lys Pro LeuGlu Arg 50 55 60 Ala Ala Phe Tyr Phe Lys Glu Ala Leu Asn Asn Leu Leu HisAsn Val 65 70 75 80 Ser Gln Thr Leu Asn Pro Tyr Ser Leu Ile Phe Lys IleAla Ala Tyr 85 90 95 Lys Ser Phe Ser Glu Ile Ser Pro Val Leu Gln Phe AlaAsn Phe Thr 100 105 110 Ser Asn Gln Ala Leu Leu Glu Ser Phe His Gly PheHis Arg Leu His 115 120 125 Ile Ile Asp Phe Asp Ile Gly Tyr Gly Gly GlnTrp Ala Ser Leu Met 130 135 140 Gln Glu Leu Val Leu Arg Asp Asn Ala AlaPro Leu Ser Leu Lys Ile 145 150 155 160 Thr Val Phe Ala Ser Pro Ala AsnHis Asp Gln Leu Glu Leu Gly Phe 165 170 175 Thr Gln Asp Asn Leu Lys HisPhe Ala Ser Glu Ile Asn Ile Ser Leu 180 185 190 Asp Ile Gln Val Leu SerLeu Asp Leu Leu Gly Ser Ile Ser Trp Pro 195 200 205 Asn Ser Ser Glu LysGlu Ala Val Ala Val Asn Ile Ser Ala Ala Ser 210 215 220 Phe Ser His LeuPro Leu Val Leu Arg Phe Val Lys His Leu Ser Pro 225 230 235 240 Thr IleIle Val Cys Ser Asp Arg Gly Cys Glu Arg Thr Asp Leu Pro 245 250 255 PheSer Gln Gln Leu Ala His Ser Leu His Ser His Thr Ala Leu Phe 260 265 270Glu Ser Leu Asp Ala Val Asn Ala Asn Leu Asp Ala Met Gln Lys Ile 275 280285 Glu Arg Phe Leu Ile Gln Pro Glu Ile Glu Lys Leu Val Leu Asp Arg 290295 300 Ser Arg Pro Ile Glu Arg Pro Met Met Thr Trp Gln Ala Met Phe Leu305 310 315 320 Gln Met Gly Phe Ser Pro Val Thr His Ser Asn Phe Thr GluSer Gln 325 330 335 Ala Glu Cys Leu Val Gln Arg Thr Pro Val Arg Gly PheHis Val Glu 340 345 350 Lys Lys His Asn Ser Leu Leu Leu Cys Trp Gln ArgThr Glu Leu Val 355 360 365 Gly Val Ser Ala Trp Arg Cys Arg Ser Ser Xaa370 375 47 745 DNA Plant 47 tgcatacaac gcaccgtttt tcgtaacacg gtttcgcgaagctctatttc atttctcctc 60 gatttttgac atgcttgaga caattgtgcc acgagaagacgaagagagga tgttccttga 120 gatggaggtc tttgggagag aggcactgaa tgtgattgcttgcgaaggtt gggaaagagt 180 ggagaggcct gagacataca agcagtggca cgtacgggctatgaggtcag ggttggtgca 240 ggttccattt gacccaagca ttatgaagac atcgctgcataaggtccaca cattctacca 300 caaggatttt gtgatcgatc aagataaccg gtggctcttgcaaggctgga agggaagaac 360 tgtcatggct ctttctgttt ggaaaccaga gtccaaggcttgaccgagaa atcctcgttg 420 gcatatgaga gaccatctct tgattttctt cctgtgtaattcccagagac agaattacag 480 atgtaagaag agaatgctgc acaaagaact tgttcaaagataatattgat gtaagtcctg 540 ttttataact ttctagctgt gtttttgttg tttctcagctagattctcct aacggtattc 600 ttgtagctag ggtgatcaga ttgtttgtat attgctagcagagttagttt gtctagattg 660 taacacatat aagaggaagc ttagagtttc tatggtttaaagagaagttt tttccttctc 720 caatgtaaaa aaaaaaaaaa aaaaa 745 48 134 PRTPlant 48 Ala Tyr Asn Ala Pro Phe Phe Val Thr Arg Phe Arg Glu Ala Leu Phe1 5 10 15 His Phe Ser Ser Ile Phe Asp Met Leu Glu Thr Ile Val Pro ArgGlu 20 25 30 Asp Glu Glu Arg Met Phe Leu Glu Met Glu Val Phe Gly Arg GluAla 35 40 45 Leu Asn Val Ile Ala Cys Glu Gly Trp Glu Arg Val Glu Arg ProGlu 50 55 60 Thr Tyr Lys Gln Trp His Val Arg Ala Met Arg Ser Gly Leu ValGln 65 70 75 80 Val Pro Phe Asp Pro Ser Ile Met Lys Thr Ser Leu His LysVal His 85 90 95 Thr Phe Tyr His Lys Asp Phe Val Ile Asp Gln Asp Asn ArgTrp Leu 100 105 110 Leu Gln Gly Trp Lys Gly Arg Thr Val Met Ala Leu SerVal Trp Lys 115 120 125 Pro Glu Ser Lys Ala Xaa 130 49 775 DNA Plant 49aaaaaatggg aaaccatcac tcttgatgaa cttatgatca atccaggaga gacaacggtc 60gtcaactgca ttcatcggtt acaatacact cctgatgaaa ctgtgtcatt agactctcca 120agagacacgg ttctgaagct attcagagat atcaatcctg acctctttgt gtttgcagag 180attaacggaa tgtacaactc tcctttcttc atgacgaggt tccgagaagc gctttttcat 240tactcttcac tctttgacat gtttgacacc acaatacacg cagaggatga gtacaaaaac 300aggtcactgt tggagagaga gttacttgtg agagacgcga tgagcgtgat ttcctgcgag 360ggtgcagagc ggtttgcgag gcctgaaacc tacaagcaat ggcgagttag gattttgaga 420gccgggttta agccagcaac tattagcaaa cagatcatga aggaggctaa ggaaattgtg 480aggaaacgtt accatagaga ttttgtgatc gatagcgata acaattggat gcttcaagga 540tggaaaggaa gagtcatcta tgctttttct tgctggaaac ctgctgagaa gttcacaaac 600aataatttaa acatctgaaa aatgttactt ctcaattaca tcatttttgt ttcccaatgg 660ttttgtagaa tatgtttgat cccgtgagtg gatgcaactc ttttttcctg caagtacata 720ttgtattcaa atccttgtgg aaatgataaa ttgtttaatc aaaaaaaaaa aaaaa 775 50 206PRT Plant 50 Lys Lys Trp Glu Thr Ile Thr Leu Asp Glu Leu Met Ile Asn ProGly 1 5 10 15 Glu Thr Thr Val Val Asn Cys Ile His Arg Leu Gln Tyr ThrPro Asp 20 25 30 Glu Thr Val Ser Leu Asp Ser Pro Arg Asp Thr Val Leu LysLeu Phe 35 40 45 Arg Asp Ile Asn Pro Asp Leu Phe Val Phe Ala Glu Ile AsnGly Met 50 55 60 Tyr Asn Ser Pro Phe Phe Met Thr Arg Phe Arg Glu Ala LeuPhe His 65 70 75 80 Tyr Ser Ser Leu Phe Asp Met Phe Asp Thr Thr Ile HisAla Glu Asp 85 90 95 Glu Tyr Lys Asn Arg Ser Leu Leu Glu Arg Glu Leu LeuVal Arg Asp 100 105 110 Ala Met Ser Val Ile Ser Cys Glu Gly Ala Glu ArgPhe Ala Arg Pro 115 120 125 Glu Thr Tyr Lys Gln Trp Arg Val Arg Ile LeuArg Ala Gly Phe Lys 130 135 140 Pro Ala Thr Ile Ser Lys Gln Ile Met LysGlu Ala Lys Glu Ile Val 145 150 155 160 Arg Lys Arg Tyr His Arg Asp PheVal Ile Asp Ser Asp Asn Asn Trp 165 170 175 Met Leu Gln Gly Trp Lys GlyArg Val Ile Tyr Ala Phe Ser Cys Trp 180 185 190 Lys Pro Ala Glu Lys PheThr Asn Asn Asn Leu Asn Ile Xaa 195 200 205 51 548 DNA Plant 51aatcgcttga accgaatttg gatcgagatt cgaaagaaag gctgagagtg gagagagtgc 60tgttcggtag gaggattatg gatttggtcc gatcagatga tgataataat aaaccgggaa 120cccggtttgg gttaatggag gagaaagaac aatggagagt gttgatggag aaagctggat 180ttgagccggt taaaccgagt aattacgcgg ttagccaagc gaagctgcta ctatggaact 240acaattatag tacattgtat tcacttgttg aatcggagcc aggtttcatc tccttggctt 300ggaacaatgt gcctctcctc accgtttcct cttggcgttg actacttggt ccgataagtt 360aatctagtat tttgagttag cttttagaat tgaattgttt ggggttagat ttggatgttt 420aattagtctc tagcctattc tcttactctt ttttgtctag tgcttggagt gatgatggtt 480tgtcgtttat gttcatttgt aatatatatt gtatgtaaca tttgactaaa aaaaaaaaaa 540aaaaaaaa 548 52 113 PRT Plant 52 Ser Leu Glu Pro Asn Leu Asp Arg Asp SerLys Glu Arg Leu Arg Val 1 5 10 15 Glu Arg Val Leu Phe Gly Arg Arg IleMet Asp Leu Val Arg Ser Asp 20 25 30 Asp Asp Asn Asn Lys Pro Gly Thr ArgPhe Gly Leu Met Glu Glu Lys 35 40 45 Glu Gln Trp Arg Val Leu Met Glu LysAla Gly Phe Glu Pro Val Lys 50 55 60 Pro Ser Asn Tyr Ala Val Ser Gln AlaLys Leu Leu Leu Trp Asn Tyr 65 70 75 80 Asn Tyr Ser Thr Leu Tyr Ser LeuVal Glu Ser Glu Pro Gly Phe Ile 85 90 95 Ser Leu Ala Trp Asn Asn Val ProLeu Leu Thr Val Ser Ser Trp Arg 100 105 110 Xaa 53 1093 DNA Plant 53gcgaatgttg agatcttgga agcaatagct ggggaaacca gagtccacat tatcgatttt 60cagattgcac agggatcaca atacatgttt ttgattcagg agcttgcgaa acgccctggt 120gggccgccgt tgctgcgtgt gacgggtgtg gatgattcac agtccaccta tgctcgtggg 180ggaggactca gcttggtagg tgagaggctt gcaactttgg cgcagtcatg tggtgtcccg 240tttgagtttc acgatgccat catgtctggg tgcaaggtgc agcgggaaca tctcgggttg 300gaacctggct ttgctgttgt tgtgaacttc ccatatgtat tacaccacat gccagacgag 360agcgtaagtg ttgaaaaata cagagacagg ctgctgcatc tgatcaagag cctctcccca 420aaactggtta ctctagtaga gcaagaatcc aacacaaaca cctcgccatt ggtgtcacgg 480tttgtggaaa cactggatta ctacacagcg atgtttgagt cgatagatgc agcacggcca 540cgggatgata agcagagaat cagcgcagaa caacactgtg tagcaagaga catagtgaac 600atgatagcat gtgaggagtc agagagagta gagagacacg aggtactggg gaaatggagg 660gtcagaatga tgatggctgg gttcacgggt tggccggtca gcacatctgc agcgtttgca 720gcgagtgaga tgctgaaagc ttatgacaaa aactacaaac tgggaggcca tgaaggagcg 780ctctacctct tctggaagag acgacccatg gctacatgtt ccgtgtggaa gccaaaccca 840aactatattg ggtaagttat agtgatgatg gttacttgag tggataaaga agagcacaac 900aaaaacacat ctgtcgctgt aaatttttta ggatgtgcaa tgatgtttta agttgtaaca 960caacctaagt tatatatgta tacaaaccaa acctggtggt tgtttttctc ttgtaaattg 1020tcatgtggtt gtgggtggga agctagtaat gaaatataac caaaacattg attaggtcaa 1080aaaaaaaaaa aaa 1093 54 285 PRT Plant 54 Ala Asn Val Glu Ile Leu Glu AlaIle Ala Gly Glu Thr Arg Val His 1 5 10 15 Ile Ile Asp Phe Gln Ile AlaGln Gly Ser Gln Tyr Met Phe Leu Ile 20 25 30 Gln Glu Leu Ala Lys Arg ProGly Gly Pro Pro Leu Leu Arg Val Thr 35 40 45 Gly Val Asp Asp Ser Gln SerThr Tyr Ala Arg Gly Gly Gly Leu Ser 50 55 60 Leu Val Gly Glu Arg Leu AlaThr Leu Ala Gln Ser Cys Gly Val Pro 65 70 75 80 Phe Glu Phe His Asp AlaIle Met Ser Gly Cys Lys Val Gln Arg Glu 85 90 95 His Leu Gly Leu Glu ProGly Phe Ala Val Val Val Asn Phe Pro Tyr 100 105 110 Val Leu His His MetPro Asp Glu Ser Val Ser Val Glu Lys Tyr Arg 115 120 125 Asp Arg Leu LeuHis Leu Ile Lys Ser Leu Ser Pro Lys Leu Val Thr 130 135 140 Leu Val GluGln Glu Ser Asn Thr Asn Thr Ser Pro Leu Val Ser Arg 145 150 155 160 PheVal Glu Thr Leu Asp Tyr Tyr Thr Ala Met Phe Glu Ser Ile Asp 165 170 175Ala Ala Arg Pro Arg Asp Asp Lys Gln Arg Ile Ser Ala Glu Gln His 180 185190 Cys Val Ala Arg Asp Ile Val Asn Met Ile Ala Cys Glu Glu Ser Glu 195200 205 Arg Val Glu Arg His Glu Val Leu Gly Lys Trp Arg Val Arg Met Met210 215 220 Met Ala Gly Phe Thr Gly Trp Pro Val Ser Thr Ser Ala Ala PheAla 225 230 235 240 Ala Ser Glu Met Leu Lys Ala Tyr Asp Lys Asn Tyr LysLeu Gly Gly 245 250 255 His Glu Gly Ala Leu Tyr Leu Phe Trp Lys Arg ArgPro Met Ala Thr 260 265 270 Cys Ser Val Trp Lys Pro Asn Pro Asn Tyr IleGly Xaa 275 280 285 55 1928 DNA Plant 55 aaagacttta gcagattttcaagcggctca gaacatcaac aacaacaaca acaacaaccg 60 ttttatagtc aagcagctctcaacgctttt ctttcaaggt ctgtgaagcc tcgaaattat 120 cagaattttc aatctccgtcggccgatgat tgatctcacg tcggtgaatg atatgagttt 180 gtttggtggt tctggttcatctcagcgtta cggtttaccg gttcccaggt ctcagacgca 240 acagcaacaa tcggattacggtttatttgg tgggatccga atgggaatcg ggtcgggtat 300 taataattat ccaacattaaccggcgttcc gtgtattgaa ccggttcaaa accgggttca 360 tgaatcggag aacatgttgaatagtttaag agagcttgag aaacagcttt tagatgatga 420 cgatgagagt ggtggtgatgatgacgtgtc agttataaca aattcaaatt ccgattggat 480 tcaaaatctc gtgactccgaacccgaaccc gaacccggtt ttgtcttttt caccgagctc 540 ttcttcttcg tcttcttcgccttctacagc ttcgacgacg acatcggtat gttctaggca 600 aacggttatg gaaatcgcgacggcgatcgc ggaagggaaa acagagatag cgacggagat 660 tttggcgcgt gtttctcaaacgcctaatct tgagaggaat tcagaggaga agcttgttga 720 tttcatggtg gctgcgcttcgatcgaggat agcttctcca gtgacggaat tgtatgggaa 780 ggagcattta atctcgactcaattgctcta cgagctctct ccttgtttca aactcggttt 840 cgaggccgcg aatctcgccattctcgacgc cgccgataac aacgacggtg gaatgatgat 900 accgcacgtt atcgatttcgatatcggaga aggtggacaa tacgttaacc ttctccgtac 960 attatccacg cgccggaatggtaaaagtca gagtcagaat tctccggtgg ttaagatcac 1020 cgccgtggcg aacaacgtttacggatgttt agtcgatgac ggtggagaag agaggttaaa 1080 agccgtcgga gatttgttgagccaactcgg tgatcgactc ggtatctccg taagtttcaa 1140 cgtggtgacg agtttacgactcggtgatct gaatcgtgaa tctctcgggt gtgatcccga 1200 cgagactttg gctgtgaacttagctttcaa gctttatcgt gttcccgacg aaagcgtatg 1260 cacggagaat ccaagagacgaacttctccg gcgcgtgaag ggacttaaac cgcgcgtggt 1320 tactctagtg gagcaagaaatgaattcgaa tacggcgccg tttttaggga gagtgagtga 1380 gtcatgcgcg tgttacggtgcgttgcttga gtcggtcgag tctacggttc ctagtacgaa 1440 ttccgaccgt gccaaagttgaggaaggaat tggccggaag ctagtaaacg cggtggcgtg 1500 cgaaggaatc gatcgtatagagcggtgcga ggtgttcggg aaatggcgaa tgcggatgag 1560 catggctggg tttgagttaatgccattgag tgagaagata gcggagtcga tgaagagtcg 1620 tggaaaccga gtccacccgggctttaccgt taaagaagat aacggaggtg tgtgctttgg 1680 ttggatggga cgggcactcactgtcgcatc cgcttggcgt taacttcaca cactcttttt 1740 tttcttctta ttattaccatattattatta attttcgaga ttattctgat attattatca 1800 ttgtgatttt ccgtttcgaaaagtgtagga atcttatgta acaaagaaaa aaaaaagact 1860 tttatgtttt tctaataataaaagaaagag tgattgggtt caaaaaaaaa aaaaaaaaaa 1920 aaaaaaaa 1928 56 524PRT Plant 56 Asp Leu Thr Ser Val Asn Asp Met Ser Leu Phe Gly Gly Ser GlySer 1 5 10 15 Ser Gln Arg Tyr Gly Leu Pro Val Pro Arg Ser Gln Thr GlnGln Gln 20 25 30 Gln Ser Asp Tyr Gly Leu Phe Gly Gly Ile Arg Met Gly IleGly Ser 35 40 45 Gly Ile Asn Asn Tyr Pro Thr Leu Thr Gly Val Pro Cys IleGlu Pro 50 55 60 Val Gln Asn Arg Val His Glu Ser Glu Asn Met Leu Asn SerLeu Arg 65 70 75 80 Glu Leu Glu Lys Gln Leu Leu Asp Asp Asp Asp Glu SerGly Gly Asp 85 90 95 Asp Asp Val Ser Val Ile Thr Asn Ser Asn Ser Asp TrpIle Gln Asn 100 105 110 Leu Val Thr Pro Asn Pro Asn Pro Asn Pro Val LeuSer Phe Ser Pro 115 120 125 Ser Ser Ser Ser Ser Ser Ser Ser Pro Ser ThrAla Ser Thr Thr Thr 130 135 140 Ser Val Cys Ser Arg Gln Thr Val Met GluIle Ala Thr Ala Ile Ala 145 150 155 160 Glu Gly Lys Thr Glu Ile Ala ThrGlu Ile Leu Ala Arg Val Ser Gln 165 170 175 Thr Pro Asn Leu Glu Arg AsnSer Glu Glu Lys Leu Val Asp Phe Met 180 185 190 Val Ala Ala Leu Arg SerArg Ile Ala Ser Pro Val Thr Glu Leu Tyr 195 200 205 Gly Lys Glu His LeuIle Ser Thr Gln Leu Leu Tyr Glu Leu Ser Pro 210 215 220 Cys Phe Lys LeuGly Phe Glu Ala Ala Asn Leu Ala Ile Leu Asp Ala 225 230 235 240 Ala AspAsn Asn Asp Gly Gly Met Met Ile Pro His Val Ile Asp Phe 245 250 255 AspIle Gly Glu Gly Gly Gln Tyr Val Asn Leu Leu Arg Thr Leu Ser 260 265 270Thr Arg Arg Asn Gly Lys Ser Gln Ser Gln Asn Ser Pro Val Val Lys 275 280285 Ile Thr Ala Val Ala Asn Asn Val Tyr Gly Cys Leu Val Asp Asp Gly 290295 300 Gly Glu Glu Arg Leu Lys Ala Val Gly Asp Leu Leu Ser Gln Leu Gly305 310 315 320 Asp Arg Leu Gly Ile Ser Val Ser Phe Asn Val Val Thr SerLeu Arg 325 330 335 Leu Gly Asp Leu Asn Arg Glu Ser Leu Gly Cys Asp ProAsp Glu Thr 340 345 350 Leu Ala Val Asn Leu Ala Phe Lys Leu Tyr Arg ValPro Asp Glu Ser 355 360 365 Val Cys Thr Glu Asn Pro Arg Asp Glu Leu LeuArg Arg Val Lys Gly 370 375 380 Leu Lys Pro Arg Val Val Thr Leu Val GluGln Glu Met Asn Ser Asn 385 390 395 400 Thr Ala Pro Phe Leu Gly Arg ValSer Glu Ser Cys Ala Cys Tyr Gly 405 410 415 Ala Leu Leu Glu Ser Val GluSer Thr Val Pro Ser Thr Asn Ser Asp 420 425 430 Arg Ala Lys Val Glu GluGly Ile Gly Arg Lys Leu Val Asn Ala Val 435 440 445 Ala Cys Glu Gly IleAsp Arg Ile Glu Arg Cys Glu Val Phe Gly Lys 450 455 460 Trp Arg Met ArgMet Ser Met Ala Gly Phe Glu Leu Met Pro Leu Ser 465 470 475 480 Glu LysIle Ala Glu Ser Met Lys Ser Arg Gly Asn Arg Val His Pro 485 490 495 GlyPhe Thr Val Lys Glu Asp Asn Gly Gly Val Cys Phe Gly Trp Met 500 505 510Gly Arg Ala Leu Thr Val Ala Ser Ala Trp Arg Xaa 515 520 57 2635 DNAPlant misc_feature (1)...(2635) n = A,T,C or G 57 tcttactcaa ggttcttctttgtcatcttg ttgccgaatc cacaaagagg agaataaaga 60 ttcgaccttt attagatattaacgactctg gatttttggg tttttggagt tggatccaca 120 tgggttctta tccggatggattccctggat ccatggacga gttggatttc aataaggact 180 ttgatttgcc tccctcctcaaaccaaacct taggtttagc taatgggttc tatttagatg 240 acttagattt ctcatccttggatcctccag aggcatatcc ctcccagaac aacaacaaca 300 acaacatcaa caacaaagctgtagcaggag atctgttatc atcttcatct gatgacgctg 360 atttctctga ttctgttttgaagtatataa gccaagttct tatggaagag gatatggaag 420 agaagccttg tatgtttcatgatgctttgg ctcttcaagc tgctgagaaa tctctctatg 480 aggctcttgg tgagaaagacccttcttcgt cttctgcttc ttctgtggat catcctgaga 540 gattggctag tcatagccctgacggttctt gttcaggtgg tgcttttagt gattacgcta 600 gcaccactac cactacttcctctgattctc actggagtgt tgatggtttg gagaatagac 660 cttcttggtt acatacacctatgccgagta attttgtttt ccagtctact tctaggtcca 720 acagtgtcac cggtggtggtggtggtggta atagtgcggt ttacggttca ggttttggcg 780 atgatttggt ttcgaatatgtttaaagatg atgaattggc tatgcagttc aagaaagggg 840 ttgaggaagc tagtaagttccttcctaagt cttctcagct ctttattgat gtggatagtt 900 acatccctat gaattctggttccaaggaaa atggttctga ggtttttgtt aagacggaga 960 agaaagatga gacagagcatcatcatcatc atagctatgc accaccaccc aacagattaa 1020 ctggtaagaa aagccattggcgcgacgaag atgaagattt cgttgaagaa agaagtaaca 1080 agcaatcagc tgtttatgttgaggaaagcg agctttctga aatgtttgat aacatgttcc 1140 tatgtggccc tgggaaacctgtatgcattc ttaaccagaa ctttcctaca gaatccgcta 1200 aagtcgtgac cgcacagtcaaatggagcaa agattcgtgg gaagaaatca acttctacta 1260 gtcatagtaa cgattctaagaaagaaactg ctgatttgag gactcttttg gtgttatgtg 1320 cacaagctgt atcagtggatgatcgtagaa ccgccaacgt ttagctaagg cagatacgag 1380 agcattcttc gcctctaggcaatggttcag agcggttggc tcattatttt gcaaatagtc 1440 ttgaagcacg cttagctgggaccggtacac agatctacac cgctttatct tcgaagaaaa 1500 cgtctgcagc agacatgttgaaggcttacc agacatacat gtcggtctgc cctttcaaga 1560 aagctgctat catatttgctaaccacagca tgatgcgttt cactgcaaac gccaacacga 1620 tccacataat agatttcggaatatcttacg gttttcagtg gcctgctctg attcatcgcc 1680 tctcgctcag cagacctggtggttcgccta agcttcgaat taccggtnnn nnnnnnnnnn 1740 nnnnnnnnnn nnnnnnnnnnnnngagttca ggagacaggt catcgcttgg ctcgatactg 1800 tcagcgacac aatgttccgtttgagtacaa cgcaattgct cagaaatggg gaaacgatcc 1860 aagtcgaaga cttaaagcttcgacaaggag agtatgtggt tgtgaactct ttgttccgtt 1920 tcaggaacct tctagatgagaccgttctgg taaacagccc gagagatgca gttttgaagc 1980 tgataagaaa aataaacccgaatgtcttca ttccagcgat cttaagcggg aattacaacg 2040 cgccattctt tgtcacgaggttcagagaag cgttgtttca ttactcggct gtgtttgata 2100 tgtgtgactc gaagctagctagggaagacg agatgaggct gatgtatgtg tttgagtttt 2160 atgggagaga gattgtgaatgttgtggctt ctgaaggaac agagagagtg gagagccgag 2220 agacatataa gcagtggcaggcgagactga tccgagccgg atttagacag cttccgcttg 2280 agaaggaact gatgcagaatctgaagttga aaatcgaaaa cgggtacgat aaaaacttcg 2340 atgttgatca aaacggtaactggttacttc aagggtggaa aggtagaatc gtgtatgctt 2400 catctctatg ggttccttcgtcttcataga tgttgtttct tacgttctaa gcgactggga 2460 tttatgtagg gcttttctgttgatagtctc tcgccaacac gagtggatta agttcagagt 2520 tagggttctt gaacactagaatgttgttat attatgcttg tgacatagcg tgtgtaagag 2580 tgtagcctaa gagatatagtactcattgca tgatcttttg ctatatgttn catgt 2635 58 809 PRT Plant VARIANT(1)...(809) Xaa = Any Amino Acid 58 Leu Leu Lys Val Leu Leu Cys His LeuVal Ala Glu Ser Thr Lys Arg 1 5 10 15 Arg Ile Lys Ile Arg Pro Leu LeuAsp Ile Asn Asp Ser Gly Phe Leu 20 25 30 Gly Phe Trp Ser Trp Ile His MetGly Ser Tyr Pro Asp Gly Phe Pro 35 40 45 Gly Ser Met Asp Glu Leu Asp PheAsn Lys Asp Phe Asp Leu Pro Pro 50 55 60 Ser Ser Asn Gln Thr Leu Gly LeuAla Asn Gly Phe Tyr Leu Asp Asp 65 70 75 80 Leu Asp Phe Ser Ser Leu AspPro Pro Glu Ala Tyr Pro Ser Gln Asn 85 90 95 Asn Asn Asn Asn Asn Ile AsnAsn Lys Ala Val Ala Gly Asp Leu Leu 100 105 110 Ser Ser Ser Ser Asp AspAla Asp Phe Ser Asp Ser Val Leu Lys Tyr 115 120 125 Ile Ser Gln Val LeuMet Glu Glu Asp Met Glu Glu Lys Pro Cys Met 130 135 140 Phe His Asp AlaLeu Ala Leu Gln Ala Ala Glu Lys Ser Leu Tyr Glu 145 150 155 160 Ala LeuGly Glu Lys Asp Pro Ser Ser Ser Ser Ala Ser Ser Val Asp 165 170 175 HisPro Glu Arg Leu Ala Ser His Ser Pro Asp Gly Ser Cys Ser Gly 180 185 190Gly Ala Phe Ser Asp Tyr Ala Ser Thr Thr Thr Thr Thr Ser Ser Asp 195 200205 Ser His Trp Ser Val Asp Gly Leu Glu Asn Arg Pro Ser Trp Leu His 210215 220 Thr Pro Met Pro Ser Asn Phe Val Phe Gln Ser Thr Ser Arg Ser Asn225 230 235 240 Ser Val Thr Gly Gly Gly Gly Gly Gly Asn Ser Ala Val TyrGly Ser 245 250 255 Gly Phe Gly Asp Asp Leu Val Ser Asn Met Phe Lys AspAsp Glu Leu 260 265 270 Ala Met Gln Phe Lys Lys Gly Val Glu Glu Ala SerLys Phe Leu Pro 275 280 285 Lys Ser Ser Gln Leu Phe Ile Asp Val Asp SerTyr Ile Pro Met Asn 290 295 300 Ser Gly Ser Lys Glu Asn Gly Ser Glu ValPhe Val Lys Thr Glu Lys 305 310 315 320 Lys Asp Glu Thr Glu His His HisHis His Ser Tyr Ala Pro Pro Pro 325 330 335 Asn Arg Leu Thr Gly Lys LysSer His Trp Arg Asp Glu Asp Glu Asp 340 345 350 Phe Val Glu Glu Arg SerAsn Lys Gln Ser Ala Val Tyr Val Glu Glu 355 360 365 Ser Glu Leu Ser GluMet Phe Asp Asn Met Phe Leu Cys Gly Pro Gly 370 375 380 Lys Pro Val CysIle Leu Asn Gln Asn Phe Pro Thr Glu Ser Ala Lys 385 390 395 400 Val ValThr Ala Gln Ser Asn Gly Ala Lys Ile Arg Gly Lys Lys Ser 405 410 415 ThrSer Thr Ser His Ser Asn Asp Ser Lys Lys Glu Thr Ala Asp Leu 420 425 430Arg Thr Leu Leu Val Leu Cys Ala Gln Ala Val Ser Val Asp Asp Arg 435 440445 Arg Thr Ala Asn Val Xaa Leu Arg Gln Ile Arg Glu His Ser Ser Pro 450455 460 Leu Gly Asn Gly Ser Glu Arg Leu Ala His Tyr Phe Ala Asn Ser Leu465 470 475 480 Glu Ala Arg Leu Ala Gly Thr Gly Thr Gln Ile Tyr Thr AlaLeu Ser 485 490 495 Ser Lys Lys Thr Ser Ala Ala Asp Met Leu Lys Ala TyrGln Thr Tyr 500 505 510 Met Ser Val Cys Pro Phe Lys Lys Ala Ala Ile IlePhe Ala Asn His 515 520 525 Ser Met Met Arg Phe Thr Ala Asn Ala Asn ThrIle His Ile Ile Asp 530 535 540 Phe Gly Ile Ser Tyr Gly Phe Gln Trp ProAla Leu Ile His Arg Leu 545 550 555 560 Ser Leu Ser Arg Pro Gly Gly SerPro Lys Leu Arg Ile Thr Gly Xaa 565 570 575 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Glu Phe Arg Arg Gln 580 585 590 Val Ile Ala Trp Leu AspThr Val Ser Asp Thr Met Phe Arg Leu Ser 595 600 605 Thr Thr Gln Leu LeuArg Asn Gly Glu Thr Ile Gln Val Glu Asp Leu 610 615 620 Lys Leu Arg GlnGly Glu Tyr Val Val Val Asn Ser Leu Phe Arg Phe 625 630 635 640 Arg AsnLeu Leu Asp Glu Thr Val Leu Val Asn Ser Pro Arg Asp Ala 645 650 655 ValLeu Lys Leu Ile Arg Lys Ile Asn Pro Asn Val Phe Ile Pro Ala 660 665 670Ile Leu Ser Gly Asn Tyr Asn Ala Pro Phe Phe Val Thr Arg Phe Arg 675 680685 Glu Ala Leu Phe His Tyr Ser Ala Val Phe Asp Met Cys Asp Ser Lys 690695 700 Leu Ala Arg Glu Asp Glu Met Arg Leu Met Tyr Val Phe Glu Phe Tyr705 710 715 720 Gly Arg Glu Ile Val Asn Val Val Ala Ser Glu Gly Thr GluArg Val 725 730 735 Glu Ser Arg Glu Thr Tyr Lys Gln Trp Gln Ala Arg LeuIle Arg Ala 740 745 750 Gly Phe Arg Gln Leu Pro Leu Glu Lys Glu Leu MetGln Asn Leu Lys 755 760 765 Leu Lys Ile Glu Asn Gly Tyr Asp Lys Asn PheAsp Val Asp Gln Asn 770 775 780 Gly Asn Trp Leu Leu Gln Gly Trp Lys GlyArg Ile Val Tyr Ala Ser 785 790 795 800 Ser Leu Trp Val Pro Ser Ser SerXaa 805 59 90 PRT Plant VARIANT (1)...(90) Xaa = Any Amino Acid 59 GlnGlu Ala Asp His Asn Lys Thr Gly Phe Leu Asp Arg Phe Thr Glu 1 5 10 15Ala Leu Phe Tyr Tyr Ser Ala Val Phe Asp Ser Leu Asp Ala Ala Asn 20 25 30Asn Asn Asn Asn Asn Asn Asn Gln Arg Met Glu Ala Glu Tyr Leu Gln 35 40 45Arg Glu Ile Cys Asp Ile Val Cys Gly Glu Gly Ala Ala Arg Xaa Glu 50 55 60Arg His Glu Pro Leu Ser Arg Trp Arg Asp Arg Leu Thr Arg Ala Gly 65 70 7580 Leu Ser Ala Val Pro Leu Gly Ser Asn Ala 85 90 60 199 DNA Plantmisc_feature (1)...(199) n = A,T,C or G 60 tctgcagaca attttnaggaggccaatacc atgctattgg aaatttcaga actgtccaca 60 cctnnnnnnn nnnnnnnnnnnnnnnnnnnn nnngtacttc tcagaggnaa tgtcggnnag 120 attagttagc tcctgcttaggaatctatgc ttctcttccn gcaacagtgg tgcctcctca 180 tggtcagaaa gtggcctca 19961 66 PRT Plant VARIANT (1)...(66) Xaa = Any Amino Acid 61 Ser Ala AspAsn Phe Xaa Glu Ala Asn Thr Met Leu Leu Glu Ile Ser 1 5 10 15 Glu LeuSer Thr Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr 20 25 30 Phe SerGlu Xaa Met Ser Xaa Arg Leu Val Ser Ser Cys Leu Gly Ile 35 40 45 Tyr AlaSer Leu Pro Ala Thr Val Val Pro Pro His Gly Gln Lys Val 50 55 60 Ala Ser65 62 321 DNA Plant misc_feature (1)...(321) n = A,T,C or G 62tcaactgaga atctagaaga tgccaacaag atgcttctgg agatttctca gttatcaaca 60ccgttcnnca cttcagcaca gcgtgtggca gcatatttct cagaagccat atcagcaagg 120ttggtgagtt catgtctagg gatatacgca actttgccac acacacacca aagccacaag 180gtagcttcag cttttcaagt gttcaatggt attagtcctt tagtggagtt ctcacacttc 240acagcaaacc aagcaattca agaagccttc gaaagagaag agagggtgca catcatagat 300cttgatataa tgcaagggtt g 321 63 107 PRT Plant VARIANT (1)...(107) Xaa =Any Amino Acid 63 Ser Thr Glu Asn Leu Glu Asp Ala Asn Lys Met Leu LeuGlu Ile Ser 1 5 10 15 Gln Leu Ser Thr Pro Phe Xaa Thr Ser Ala Gln ArgVal Ala Ala Tyr 20 25 30 Phe Ser Glu Ala Ile Ser Ala Arg Leu Val Ser SerCys Leu Gly Ile 35 40 45 Tyr Ala Thr Leu Pro His Thr His Gln Ser His LysVal Ala Ser Ala 50 55 60 Phe Gln Val Phe Asn Gly Ile Ser Pro Leu Val GluPhe Ser His Phe 65 70 75 80 Thr Ala Asn Gln Ala Ile Gln Glu Ala Phe GluArg Glu Glu Arg Val 85 90 95 His Ile Ile Asp Leu Asp Ile Met Gln Gly Leu100 105 64 195 DNA Plant misc_feature (1)...(195) n = A,T,C or G 64tctgcagaca actttgaaga agccaataca atactgcctc agatcacaga actctccacc 60ccctatngca actcggtgca acgagtggct gcctatnnnn nnnnnnnnnn nnnnnnnnnn 120nnnnnnnnnn nntgcatagg aatgtattct cctctccctc ctattcacat gtcccagagc 180cagaaaattg tgaat 195 65 65 PRT Plant VARIANT (1)...(65) Xaa = Any AminoAcid 65 Ser Ala Asp Asn Phe Glu Glu Ala Asn Thr Ile Leu Pro Gln Ile Thr1 5 10 15 Glu Leu Ser Thr Pro Tyr Xaa Asn Ser Val Gln Arg Val Ala AlaTyr 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Ile GlyMet 35 40 45 Tyr Ser Pro Leu Pro Pro Ile His Met Ser Gln Ser Gln Lys IleVal 50 55 60 Asn 65 66 2151 DNA Plant 66 gatatcagca tcatcaattttaaatgtaag ttggcaaaag atcatgaggg ttctcatagt 60 aatttggcca caaggtatgacactgtctca attgagcaat ctagtagaga aactgatcca 120 tcatatattg ctcatattgaaagtgaaaaa gatatgctca agaacctagt agagaagcta 180 aaaattgaaa aatctagctctactagaaaa atatgatagg ttgcctgttt ctcatgaaaa 240 tttattagat aatcatatcatggctagatg tcgctcatga ggttgttctt gctagtttag 300 attcctgtgg gcattcatctcttttagatg cactaacatg ataggaagtt tctaatctgg 360 tgcttcacaa ttctggtgattcatgcttcc ttcattgcaa ttgatattga tgcttgattc 420 atgcttcagt cactttgtgcgtttaattgg tattgtatgt atcactagat tgtagggtgt 480 ctgcaactag tgtttcaccatgtggttttt tagtatcatt cgtattagtt tctaactttc 540 tattgatata ttaaagtgataactagtttt agaaatattc tcttgtgcca ttaatgctac 600 aacttgtttt tagcgtgtacgttagcatta taatatttcc ttattatgaa agcggaagag 660 aaacgcgccc aaccagagcatccacgtcgt ctcatttcac cttcatcgtt ggatcataga 720 tgagcggtcc acggtgaactccgtttgcct gcaaaaccac gtcctctacg cgctgttaag 780 tagcttctag aaacatcacgatgtgtcccg tccattcctt taggaggagc cggatccggc 840 gccgcagtcg cccaaggtcccgaccgccgc ggcctcggcc gccgccgcca aggagcggaa 900 ggaggtgcag cggcggaagcagcgcgacga ggagggcctc cacctgctga gtgctgacgc 960 tgctgctgca gtgcgcggaggccgtgaacg cggacaacct cgacgacgcg caccagacgc 1020 tgctggagat cgcggagctggccacgccgt tcggcacctc gacccagcgc gtggccgcct 1080 acttcgcgga ggccatgtcggcgcgcgtcg tcagctcctg cctaggcctg tacgcgccgc 1140 tgccgccggg ctcccccgccgcggcgcgcc tccacggccg cgtggccgcc gcgttccagg 1200 tgttcaacgg catcagccccttcgtcaagt tctcgcactt caccgccaac caggccatcc 1260 aggaggcgtt cgagcgggaggagcgtgtgc acatcatcga cctcgacatc atgcaggggc 1320 tgcagtggcc gggcctcttccacatccttg tctcccgccc cggcggcccg cccagggtca 1380 ggctcaccgg cctgggggcgtccatggacg cgctcgaggc gacggggaag cgcctctccg 1440 acttcgccga cacgctcggcctgcccttcg agttctgcgc cgtcgccgag aaggccggca 1500 acgttgaccc gcagaagctgggcgtcacgc ggcgggaggc cgtcgccgtc cactggccgc 1560 accactcgct ttacgacgtcatcggctccg actccaacac gctctggctc atccaaaggt 1620 cctccatttt ccttctctgcctttcttcca tgtcaaatct tgatgcaatc atgaccactt 1680 ttcagctgct gacattggataatgtgagct ttacggcaag catcaagtcg tggtagtaca 1740 tccattacag ctatttctaaaatattcttc ggaggtttcc tgctcatagt aaaaaaaaat 1800 cgcgttttga agctcaaaaggcgatttctt ccgaggtttg ctgttgagcg ctattttgga 1860 aaccccattt tctcaattgatttttatttt ttaaagaaaa attagttcat ttttctcttg 1920 tgaaatggag tcccaaactaaccctaatat taaaaaaaac gcgctttgga gctcaaaacg 1980 ctcgttgtta tgaccaaccagctttatagg tttaaaaagg ttgaatcttg acaatgcttt 2040 tgaaaaggtt gaatcttgacaatgcttttg agatgatact gtagtgtagt ctgtagtgga 2100 gcatcctcca tggtctttggtgatcgagaa ttcctgcagc ccgggggatc c 2151 67 716 PRT Plant VARIANT(1)...(716) Xaa = Any Amino Acid 67 Tyr Gln His His Gln Phe Xaa Met XaaVal Gly Lys Arg Ser Xaa Gly 1 5 10 15 Phe Ser Xaa Xaa Phe Gly His LysVal Xaa His Cys Leu Asn Xaa Ala 20 25 30 Ile Xaa Xaa Arg Asn Xaa Ser IleIle Tyr Cys Ser Tyr Xaa Lys Xaa 35 40 45 Lys Arg Tyr Ala Gln Glu Pro SerArg Glu Ala Lys Asn Xaa Lys Ile 50 55 60 Xaa Leu Tyr Xaa Lys Asn Met IleGly Cys Leu Phe Leu Met Lys Ile 65 70 75 80 Tyr Xaa Ile Ile Ile Ser TrpLeu Asp Val Ala His Glu Val Val Leu 85 90 95 Ala Ser Leu Asp Ser Cys GlyHis Ser Ser Leu Leu Asp Ala Leu Thr 100 105 110 Xaa Xaa Glu Val Ser AsnLeu Val Leu His Asn Ser Gly Asp Ser Cys 115 120 125 Phe Leu His Cys AsnXaa Tyr Xaa Cys Leu Ile His Ala Ser Val Thr 130 135 140 Leu Cys Val XaaLeu Val Leu Tyr Val Ser Leu Asp Cys Arg Val Ser 145 150 155 160 Ala ThrSer Val Ser Pro Cys Gly Phe Leu Val Ser Phe Val Leu Val 165 170 175 SerAsn Phe Leu Leu Ile Tyr Xaa Ser Asp Asn Xaa Phe Xaa Lys Tyr 180 185 190Ser Leu Val Pro Leu Met Leu Gln Leu Val Phe Ser Val Tyr Val Ser 195 200205 Ile Ile Ile Phe Pro Tyr Tyr Glu Ser Gly Arg Glu Thr Arg Pro Thr 210215 220 Arg Ala Ser Thr Ser Ser His Phe Thr Phe Ile Val Gly Ser Xaa Met225 230 235 240 Ser Gly Pro Arg Xaa Thr Pro Phe Ala Cys Lys Thr Thr SerSer Thr 245 250 255 Arg Cys Xaa Val Ala Ser Arg Asn Ile Thr Met Cys ProVal His Ser 260 265 270 Phe Arg Arg Ser Arg Ile Arg Arg Arg Ser Arg ProArg Ser Arg Pro 275 280 285 Pro Arg Pro Arg Pro Pro Pro Pro Arg Ser GlyArg Arg Cys Ser Gly 290 295 300 Gly Ser Ser Ala Thr Arg Arg Ala Ser ThrCys Xaa Val Leu Thr Leu 305 310 315 320 Leu Leu Gln Cys Ala Glu Ala ValAsn Ala Asp Asn Leu Asp Asp Ala 325 330 335 His Gln Thr Leu Leu Glu IleAla Glu Leu Ala Thr Pro Phe Gly Thr 340 345 350 Ser Thr Gln Arg Val AlaAla Tyr Phe Ala Glu Ala Met Ser Ala Arg 355 360 365 Val Val Ser Ser CysLeu Gly Leu Tyr Ala Pro Leu Pro Pro Gly Ser 370 375 380 Pro Ala Ala AlaArg Leu His Gly Arg Val Ala Ala Ala Phe Gln Val 385 390 395 400 Phe AsnGly Ile Ser Pro Phe Val Lys Phe Ser His Phe Thr Ala Asn 405 410 415 GlnAla Ile Gln Glu Ala Phe Glu Arg Glu Glu Arg Val His Ile Ile 420 425 430Asp Leu Asp Ile Met Gln Gly Leu Gln Trp Pro Gly Leu Phe His Ile 435 440445 Leu Val Ser Arg Pro Gly Gly Pro Pro Arg Val Arg Leu Thr Gly Leu 450455 460 Gly Ala Ser Met Asp Ala Leu Glu Ala Thr Gly Lys Arg Leu Ser Asp465 470 475 480 Phe Ala Asp Thr Leu Gly Leu Pro Phe Glu Phe Cys Ala ValAla Glu 485 490 495 Lys Ala Gly Asn Val Asp Pro Gln Lys Leu Gly Val ThrArg Arg Glu 500 505 510 Ala Val Ala Val His Trp Pro His His Ser Leu TyrAsp Val Ile Gly 515 520 525 Ser Asp Ser Asn Thr Leu Trp Leu Ile Gln ArgSer Ser Ile Phe Leu 530 535 540 Leu Cys Leu Ser Ser Met Ser Asn Leu AspAla Ile Met Thr Thr Phe 545 550 555 560 Gln Leu Leu Thr Leu Asp Asn ValSer Phe Thr Ala Ser Ile Lys Ser 565 570 575 Trp Xaa Tyr Ile His Tyr SerTyr Phe Xaa Asn Ile Leu Arg Arg Phe 580 585 590 Pro Ala His Ser Lys LysLys Ser Arg Phe Glu Ala Gln Lys Ala Ile 595 600 605 Ser Ser Glu Val CysCys Xaa Ala Leu Phe Trp Lys Pro His Phe Leu 610 615 620 Asn Xaa Phe LeuPhe Phe Lys Glu Lys Leu Val His Phe Ser Leu Val 625 630 635 640 Lys TrpSer Pro Lys Leu Thr Leu Ile Leu Lys Lys Thr Arg Phe Gly 645 650 655 AlaGln Asn Ala Arg Cys Tyr Asp Gln Pro Ala Leu Xaa Val Xaa Lys 660 665 670Gly Xaa Ile Leu Thr Met Leu Leu Lys Arg Leu Asn Leu Asp Asn Ala 675 680685 Phe Glu Met Ile Leu Xaa Cys Ser Leu Xaa Trp Ser Ile Leu His Gly 690695 700 Leu Trp Xaa Ser Arg Ile Pro Ala Ala Arg Gly Ile 705 710 715 6823 DNA Artificial Sequence CDS (1)...(23) Primer 68 cay tty acn gcn aaycar gcn at 23 69 8 PRT Artificial Sequence primer 69 His Phe Thr Ala AsnGln Ala Ile 1 5 70 29 DNA Artificial Sequence CDS (10)...(29) Primer 70acgtctcga gtn cay ath ath gay ttn ga 29 71 7 PRT Artificial SequenceVARIANT (1)...(7) Xaa = Any Amino Acid 71 Val His Ile Ile Asp Xaa Asp 15 72 20 DNA Artificial Sequence CDS (1)...(20) Primer 72 ytn car tgy gcngar gcn gt 20 73 7 PRT Artificial Sequence Primer 73 Leu Gln Cys Ala GluAla Val 1 5 74 23 DNA Artificial Sequence CDS (3)...(23) Primer 74 ckccm gtk tgg ngg ncc ncc ngg 23 75 8 PRT Artificial Sequence VARIANT(1)...(8) Xaa = Any Amino Acid 75 Pro Gly Gly Pro Pro Xaa Xaa Arg 1 5 7623 DNA Artificial Sequence CDS (3)...(23) Primer 76 at ncc rtt raa nacytg raa ngc 23 77 8 PRT Artificial Sequence Primer 77 Ala Phe Gln ValPhe Asn Gly Ile 1 5 78 23 DNA Artificial Sequence CDS (3)...(23) Primer78 at rtg raa nar ncc ngg cca ytg 23 79 8 PRT Artificial Sequence Primer79 Gln Trp Pro Gly Leu Phe His Ile 1 5

What is claimed is:
 1. An isolated nucleic acid molecule whichhybridizes over its full length to the complement of the nucleic acidmolecule of SEQ ID NO:1 under highly stringent conditions, wherein saidhighly stringent conditions consist of hybridization to filter-bound DNAin 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C.,and washing in 0.1×SSC/0.1% SDS at 68° C., wherein said isolated nucleicacid molecule encodes a SCARECROW protein that has the property of SEQID NO:2 of directing the asymmetric division of the cortex/endodermalinitial of a plant.
 2. The isolated nucleic acid molecule of claim 1,wherein said isolated nucleic acid molecule is expressed in theendodermis, cortex/endodermal initial, or quiescent center of awild-plant.
 3. An isolated nucleic acid molecule wherein the nucleicacid molecule comprises SEQ ID NO:1.
 4. An isolated nucleic acidmolecule comprising a nucleic acid sequence that encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO:2.
 5. A DNA vectorcomprising the isolated nucleic acid molecule in any one of claims 2, 1,3, or
 4. 6. An expression vector comprising the isolated nucleic acidmolecule in any one of claims 2, 1, 3, or 4, operatively associated witha regulatory sequence comprising transcriptional and translationalregulatory elements that control expression of the isolated nucleic acidmolecule in a plant host cell.
 7. A genetically-engineered plant hostcell containing the isolated nucleic acid molecule in any one of claims2, 1, 3, or
 4. 8. A genetically-engineered plant host cell containingthe isolated nucleic acid molecule in any one of claims 2, 1, 3, or 4,operatively associated with a regulatory sequence comprisingtranscriptional and translational regulatory elements that controlexpression of the isolated nucleic acid molecule in said host cell.
 9. Agenetically-engineered plant containing the isolated nucleic acidmolecule in any one of claims 2, 1, 3, or
 4. 10. A plantgenetically-engineered to overexpress a SCARECROW protein orpolypeptide, said plant transformed with the isolated nucleic acidmolecule in any one of claims 2, 1, 3 or
 4. 11. A method for expressinga nucleic acid molecule that encodes a SCARECROW protein in a a planthost cell, comprising: (a) culturing the genetically-engineered planthost cell of claim 8; and (b) inducing the transcriptional andtranslational regulatory elements that control expression of theisolated nucleic acid molecule.
 12. A method for producing a transgenicplant, comprising transforming a plant cell with the isolated nucleicacid molecule of any one of claims 2, 1, 3, or 4 operatively associatedwith a regulatory sequence comprising transcriptional and translationalregulatory elements that control expression of the isolated nucleic acidmolecule in the plant cell, and regenerating a transgenic plant from thetransformed plant cell.